Figure 3Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos ( A ) , in the CM from E14 cortical cells in culture for 4 daysWestern blot analysis for CSTB in the CM from hCOs after 4 days in culture ( C ) . The CM was collected from hCOs at different time points in culture ( d19 , d38 , d60 , d80 ) . Protein extract ( PE ) from 60 days hCOs was used as a positive control . GAPDH immunostaining was used as a negative control on hCOs CM . Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM samplesMicrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry expressing vector and HA - empty vector or - Cstb , and analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65 - GFP transgenic mouse line . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of GFP + interneurons in the 5 equal Bins of the mouse developing cortex - Bin1 corresponded to apical side and Bin5 to pial side . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry and HA - Cstb expressing vector , analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67 - GFP transgenic mouse line . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of GFP + interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro - caudally .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos (A), in the CM from E14 cortical cells in culture for 4 daysWestern blot analysis for CSTB in the CM from hCOs after 4 days in culture (C). The CM was collected from hCOs at different time points in culture (d19, d38, d60, d80). Protein extract (PE) from 60 days hCOs was used as a positive control. GAPDH immunostaining was used as a negative control on hCOs CM. Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM samplesMicrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-empty vector or -Cstb, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex-Bin1 corresponded to apical side and Bin5 to pial side. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles.Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry and HA-Cstb expressing vector, analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro-caudally.


Figure 4Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP - miR - neg vector ( CTRL ) and GFP - miRNA - Cstb ( KD ) , analyzed 3 dpe and immunostained with GFP . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of electroporated GFP + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP + cells calculated in each Bin . Nuclei ( blue ) are stained with DAPI . Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP - empty vector control and GFP - miRNA - Cstb , analyzed 3 dpe and immunostained with GFP and Ki67 . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) of ventricular structures transfected with GFP - miR - neg vector or GFP - miRNA - Cstb in ( C ) . Nuclei ( blue ) are stained with DAPI . Micrograph sections of d40 hCOs electroporated with GFP - CSTB expressing vector or GFP - R68X mutant and analyzed 5 dpe . Sections were immunostained for GFP and Ki67 . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Quantification of the total number of proliferating KI67 + cells / area ( µm2 ) ( F ) and Ki67 + GFP + cells / area ( µm2 ) ( G ) of ventricles like structures transfected with GFP - empty vector control , GFP - CSTB or GFP - R68X mutant in ( E ) . Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector control , GFP - Cstb or GFP - R68X , analyzed 2 dpe and immunostained with GFP and Phospho - Histone H3 ( PH3 ) . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of electroporated GFP + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP + cells calculated in each Bin . Distribution of mitotic progenitor PH3 + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3 + cells calculated in each Bin . K . Quantification of the total number of proliferating PH3 + cells / area ( µm2 ) of ventricular structures transfected with GFP - empty vector control , GFP - Cstb or GFP - R68X mutant in ( H ) . Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector control , GFP - Cstb or GFP - R68X mutant , analyzed 2 dpe and immunostained with GFP and Tbr2 . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of Tbr2 + cells / cortical area ( µm2 ) in ( L ) . Data shown as Z - scores relative to the mean of GFP control vector . Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry expressing vector and HA - Cstb or R68X , and analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65 - GFP transgenic mouse line . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of GAD65 - GFP interneurons / cortical area ( µm2 ) in ( N ) . Data shown as Z - scores relative to the mean of GFP control vector . Nuclei ( blue ) are stained with DAPI .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-miR-neg vector (CTRL) and GFP-miRNA-Cstb (KD), analyzed 3 dpe and immunostained with GFP. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin. Nuclei (blue) are stained with DAPI.Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-empty vector control and GFP-miRNA-Cstb, analyzed 3 dpe and immunostained with GFP and Ki67. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricular structures transfected with GFP-miR-neg vector or GFP-miRNA- Cstb in (C). Nuclei (blue) are stained with DAPI.Micrograph sections of d40 hCOs electroporated with GFP-CSTB expressing vector or GFP-R68X mutant and analyzed 5 dpe. Sections were immunostained for GFP and Ki67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area(µm2)(F)and Ki67+GFP+ cells/area(µm2)(G) of ventricles like structures transfected with GFP-empty vector control, GFP-CSTB or GFP-R68X mutant in (E).Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector control, GFP-Cstb or GFP-R68X, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin.Distribution of mitotic progenitor PH3+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells calculated in each Bin.K.Quantification of the total number of proliferating PH3+ cells/area(µm2)of ventricular structures transfected with GFP-empty vector control, GFP-Cstb or GFP-R68X mutant in (H).Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector control, GFP-Cstb or GFP-R68X mutant, analyzed 2 dpe and immunostained with GFP and Tbr2. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of Tbr2+ cells/ cortical area (µm2) in (L). Data shown as Z-scores relative to the mean of GFP control vector.Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-Cstb or R68X, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of GAD65-GFP interneurons/ cortical area (µm2) in (N). Data shown as Z-scores relative to the mean of GFP control vector. Nuclei (blue) are stained with DAPI.


Figure 5Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points . Right panel shows the difference of the areas of f - CTRL , b - CTRL , UL1 and UL4 hCOs at different time in culture . Data shown as Z - scores relative to the mean of b - CTRL . Micrograph sections of d30 b - CTRL , UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3 . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) of ventricles like structures and of the number of PH3 + cells facing the ventricle / apical surface length ( µm ) . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI . Micrograph sections of d60 b - CTRL , UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3 . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the total number of proliferating KI67 + cells / area of ventricles like structures and of the number of PH3 + cells facing the ventricle / the apical surface length ( µm ) . Data shown as Z - scores relative to the mean of CTRL . Nuclei ( blue ) are stained with DAPI . Scheme of the experiment with conditioned medium exchange between b - CTRL and UL4 hCOs and quantification of the Ki67 + cells by FACS analysis on 36 days old dissociated hCOs . Four samples were analyzed as biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points. Right panel shows the difference of the areas of f-CTRL, b-CTRL, UL1 and UL4 hCOs at different time in culture. Data shown as Z-scores relative to the mean of b-CTRL.Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricles like structures and of the number of PH3+ cells facing the ventricle/apical surface length (µm). Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating KI67+ cells/ area of ventricles like structures and of the number of PH3+ cells facing the ventricle/the apical surface length (µm). Data shown as Z-scores relative to the mean of CTRL. Nuclei (blue) are stained with DAPI.Scheme of the experiment with conditioned medium exchange between b-CTRL and UL4 hCOs and quantification of the Ki67+ cells by FACS analysis on 36 days old dissociated hCOs. Four samples were analyzed as biological replicates.


Figure 6Micrograph sections of d30 b - CTRL , UL1 and UL4 hCOs immunostained for the neuronal marker DCX . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the DCX + area . The area of DCX + cells was calculated and related to the total area ( µm2 ) of the ventricular structure . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI . Micrograph sections of d60 b - CTRL , UL1 and UL4 hCOs immunostained for neuronal marker DCX . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the DCX + area . The area ( µm2 ) of DCX + cells was calculated and related to the total area of the ventricular structure . Data shown as Z - scores relative to the mean of b - CTRL . Quantification of the number of neuronal DCX + cells by FACS sorting on 60 days old dissociated hCOs . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI . Micrograph sections of d30 fused - organoid ; ventral GFP - f - CTRL and dorsal UL1 . Micrographs of sections from d30 fused organoids , showing ventral GFP + cells in the dorsal area . The migrating GFP + cells were counted starting from 100 µm far from the ventral / dorsal border . Quantification of ventrally - derived GFP + cells in dorsal / thickness of the counted area ( µm3 ) . The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig . EV5D . Every dot represents a different hCOs and different areas were counted for each hCO . Nuclei ( blue ) are stained with DAPI . Volcano plot representing proteins identified by high - resolution nanoLC MS / MS analysis . Up - and down - regulated proteins ( - 0 . 3 ≥ log2FC ≥ 0 . 3 ) in human cerebral organoids ( UL1 vs b - CTRL ) are shown in red and green , respectively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for the neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area of DCX+ cells was calculated and related to the total area (µm2) of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area (µm2) of DCX+ cells was calculated and related to the total area of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Quantification of the number of neuronal DCX+ cells by FACS sorting on 60 days old dissociated hCOs. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.Micrograph sections of d30 fused-organoid; ventral GFP-f-CTRL and dorsal UL1.Micrographs of sections from d30 fused organoids, showing ventral GFP+ cells in the dorsal area. The migrating GFP+ cells were counted starting from 100 µm far from the ventral/dorsal border. Quantification of ventrally-derived GFP+ cells in dorsal/thickness of the counted area (µm3). The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig.EV5D. Every dot represents a different hCOs and different areas were counted for each hCO. Nuclei (blue) are stained with DAPI.Volcano plot representing proteins identified by high-resolution nanoLC MS/MS analysis. Up- and down-regulated proteins (-0.3≥log2FC≥0.3) in human cerebral organoids (UL1 vs b-CTRL) are shown in red and green, respectively.


Figure 1A - C Calcium flux measured by FACScan for splenic B cells isolated from B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( see Figure EV1 , 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . The addition of the stimuli to the cells is indicated by arrows . D Representative microscopy images showing Fab - PLA results measuring the BCR proximity for the IgM - BCR ( upper ) and the IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B cells . PLA signals are shown as red dots and the nuclei are visualized by DAPI staining . Scale bar : 5 μm . E The Fab - PLA results are quantified by Blobfinder software and presented as box plots . The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample . Data from treated samples were compared to data from resting cells , p - values were calculated by Kruskal - Wallis one - way analysis of variance ( ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-C Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (see Figure EV1, 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows.D Representative microscopy images showing Fab-PLA results measuring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots and the nuclei are visualized by DAPI staining. Scale bar: 5 μm.E The Fab-PLA results are quantified by Blobfinder software and presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from treated samples were compared to data from resting cells, p-values were calculated by Kruskal-Wallis one-way analysis of variance (ANOVA).


Figure 2A - C Calcium flux measured by FACScan for splenic B cells isolated from Lyn - deficient B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells . D Representative microscopy images showing Fab - PLA results monitoring the BCR proximity for the IgM - BCR ( upper ) and the IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B cells . Scale bar : 5 μm . E Quantified Fab - PLA results are presented as box plots , where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample . p - values were calculated by Kruskal - Wallis one - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-C Calcium flux measured by FACScan for splenic B cells isolated from Lyn-deficient B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.D Representative microscopy images showing Fab-PLA results monitoring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. Scale bar: 5 μm.E Quantified Fab-PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. p-values were calculated by Kruskal-Wallis one-way ANOVA.


Figure 3A - D Proximity between IgM - BCR on the surface of 3046SM ( A , C ) or 3046M ( B , D ) cells before and after a 1 - min stimulation with the indicated reagents , assayed by Fab - PLA . Results are presented as representative microscopy images ( A , B ) and box plots after quantification ( C , D ) . Scale bar : 5 μm . In the box plots , the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample and p - values were calculated by Kruskal - Wallis one - way ANOVA . E - H Calcium flux measured by FACScan for 3046SM ( E , F ) and 3046M ( G , H ) cells after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-D Proximity between IgM-BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1-min stimulation with the indicated reagents, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.E-H Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


Figure 4A - D Proximity between IgD - BCRs on the surface of 3046SD ( A , C ) or 3046D ( B , D ) cells before and after a 1 - min stimulation , assayed by Fab - PLA . Results are presented as representative microscopy images ( A , B ) and box plots after quantification ( C , D ) . PLA signals are shown as red dots and nuclei were visualized by DAPI staining . Scale bar : 5 μm . In the box plots , the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample and p - values were calculated by Kruskal - Wallis one - way ANOVA . E - H Calcium flux measured by FACScan for 3046SD ( E , F ) and 3046D ( G , H ) cells after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-D Proximity between IgD-BCRs on the surface of 3046SD (A, C) or 3046D (B, D) cells before and after a 1-min stimulation, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.E-H Calcium flux measured by FACScan for 3046SD (E, F) and 3046D (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


Figure 5Calcium flux measured by FACScan for 3046M ( A , D ) , 3046SD ( B , E ) and 3046D ( C , F ) cells stimulated with anti - HC ( 2 μl / ml ) ( A , B , C ) , or anti - λ ( 2 μl / ml ) ( D , E , F ) antibodies with or without 1 min prestimulation with 1NIP - pep ( 80 nM ) . The addition of antibodies is indicated by black arrows . The 1NIP - pep was added immediately before recording . Data are representative of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Calcium flux measured by FACScan for 3046M (A, D), 3046SD (B, E) and 3046D (C, F) cells stimulated with anti-HC (2 μl/ml) (A, B, C), or anti-λ (2 μl/ml) (D, E, F) antibodies with or without 1 min prestimulation with 1NIP-pep (80 nM). The addition of antibodies is indicated by black arrows. The 1NIP-pep was added immediately before recording. Data are representative of three independent experiments.


Figure 6A - E B1 - 8 B cells , 3046SM cells , 3046M cells , 3046SD cells , or 3046D cells were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD19 antibody ( 62 nM ) , or costimulated with 1NIP - pep and anti - CD19 antibody . The calcium fluxes were measured by FACScan . F - J B1 - 8 B cells , 3046SM cells , 3046M cells , 3046SD cells , or 3046D cells were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD20 antibody ( 62 nM ) , or costimulated with 1NIP - pep and anti - CD20 antibody . The calcium fluxes were measured by FACScan .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-E B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD19 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD19 antibody. The calcium fluxes were measured by FACScan.F-J B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD20 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD20 antibody. The calcium fluxes were measured by FACScan.


Figure 3 ( D ) Validation by western blot of changes in specific phosphorylation events on kinases . Left , Immunoblots of cortical lysates of CTL and micTNFα - KO at ZT6 for : ( top ) phosphorylated GSK3α / β ( pGSK3α at Ser21 and pGSK3β at Ser9 ) and total GSK3α ; and ( bottom ) phosphorylated MAPK ( pMAPK , MAPK3 at Thr203 / Tyr205 and MAPK1 at Thr183 / Tyr185 ) and total MAPK . Right , Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6 . n = 5 mice per group . * * * p = 0 . 000009 ( GSK3α ) , multiple t - test with Benjamini - Hochberg correction . ( E ) Kinases with altered activity between CTL and micTNF - KOs predicted by robust kinase activity inference ( RoKAI ) at ZT18 and ZT6 . Graph shows the z - score value of each kinase assigned by RoKAI . Kinases above and below the dotted line represent prediction of up - and downregulated activity in micTNFα - KO , respectively . Kinases are color - coded according to kinase group as described in ( C ) . ( F Validation by western blot of altered activity of predicted kinases between CTL and micTNF - KOs . ( F ) Left , Immunoblots of cortical lysates of CTL and micTNFα - KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK / CDK ( PXS * P , S * PX ( K / R ) ) and PKC ( ( K / R ) XS * X ( K / R ) ) . Dashed boxes indicate quantified signal . Right , Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6 . ( F , n = 5 mice per group . * p = 0 . 022 ( MAPK / CDK phospho sub ) , * p = 0 . 014 ( PKC phospho sub ) multiple t - test with Benjamini - Hochberg correction . G ) Validation by western blot of altered activity of predicted kinases between CTL and micTNF - KOs . ( G ) Left , Immunoblots show signal of phosphorylated threonine in MARKs activation loop . Right , Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6 . G ) n = 5 mice per group . * * p = 0 . 0091 ( MARK ) , multiple t - test with Benjamini - Hochberg correction .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(D) Validation by western blot of changes in specific phosphorylation events on kinases. Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 for: (top) phosphorylated GSK3α/β (pGSK3α at Ser21 and pGSK3β at Ser9) and total GSK3α; and (bottom) phosphorylated MAPK (pMAPK, MAPK3 at Thr203/Tyr205 and MAPK1 at Thr183/Tyr185) and total MAPK. Right, Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6. n=5 mice per group. ***p=0.000009 (GSK3α), multiple t-test with Benjamini-Hochberg correction.(E) Kinases with altered activity between CTL and micTNF-KOs predicted by robust kinase activity inference (RoKAI) at ZT18 and ZT6. Graph shows the z-score value of each kinase assigned by RoKAI. Kinases above and below the dotted line represent prediction of up- and downregulated activity in micTNFα-KO, respectively. Kinases are color-coded according to kinase group as described in (C).(F Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (F) Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK/CDK (PXS*P, S*PX(K/R)) and PKC ((K/R)XS*X(K/R)). Dashed boxes indicate quantified signal. Right, Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6. (F, n=5 mice per group. *p=0.022 (MAPK/CDK phospho sub), *p=0.014 (PKC phospho sub) multiple t-test with Benjamini-Hochberg correction.G) Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (G) Left, Immunoblots show signal of phosphorylated threonine in MARKs activation loop. Right, Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6. G) n=5 mice per group. **p=0.0091 (MARK), multiple t-test with Benjamini-Hochberg correction.


Figure 1 ( A ) 3D time ‐ lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron . Neurons were transfected with Mito ‐ DsRed2 , pretreated with the pan ‐ caspase inhibitor zVAD ‐ fmk methyl ester ( 100 μM ) , and exposed to SNOC ( 200 μM ) . Images were 3D iso ‐ surface rendered . Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure ( upper panels ; scale bar , 15 μm ) and closeup views ( lower panels ; scale bar , 3 μm ) . See Supplementary Video 1 . ( B ) Mitochondrial fragmentation within the field of view shown in ( A ) results in increased mitochondrial numbers . ( C ) NO induces dose ‐ dependent mitochondrial fission . Neurons expressing Mito ‐ DsRed2 were exposed to SNOC and fixed at 60 min . The fraction of neurons displaying fragmented mitochondria is shown as the mean ± s . e . m . ( D ) Mito ‐ DsRed2 ‐ expressing corticalneurons were pretreated with 1 mM nitro ‐ L ‐ arginine ( 20 min ) and exposed to 25 μM NMDA . The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean ± s . e . m . of quintuplicate samples from a representative experiment ( † † † significance at P0 . 001 or n . s . † , not significant compared to control ; * * * significance at P0 . 001 as compared to NMDA treatment ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) 3D time‐lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron. Neurons were transfected with Mito‐DsRed2, pretreated with the pan‐caspase inhibitor zVAD‐fmk methyl ester (100 μM), and exposed to SNOC (200 μM). Images were 3D iso‐surface rendered. Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure (upper panels; scale bar, 15 μm) and closeup views (lower panels; scale bar, 3 μm). See Supplementary Video 1.(B) Mitochondrial fragmentation within the field of view shown in (A) results in increased mitochondrial numbers.(C) NO induces dose‐dependent mitochondrial fission. Neurons expressing Mito‐DsRed2 were exposed to SNOC and fixed at 60 min. The fraction of neurons displaying fragmented mitochondria is shown as the mean±s.e.m.(D) Mito‐DsRed2‐expressing corticalneurons were pretreated with 1 mM nitro‐L‐arginine (20 min) and exposed to 25 μM NMDA. The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean±s.e.m. of quintuplicate samples from a representative experiment (†††significance at P0.001 or n.s.†, not significant compared to control; ***significance at P0.001 as compared to NMDA treatment).


Figure 2Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria . Neurons were co ‐ transfected with vectors encoding ( A ) Mito ‐ DsRed2 and ( B ) MyrPalm ‐ mCFP plus ( C ) AIF ‐ GFP and the effects of SNOC were recorded using 3D time ‐ lapse imaging . Projection images zoomed in onto the dendritic arbor of one neuron ( representative of n = 42 ) . Arrows indicate dendritic spines . The inset shows the soma at appropriate grayscale levels . The apparent brightening of AIF ‐ GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF . Scale bars , 10 μm . See also Supplementary Video 2 . ( D ) 3D time ‐ lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition . Scale bar , 10 μm . Five mitochondria were selected and motion tracked throughout the imaging series . ( E ) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion . Each trace color corresponds to the same colored mitochondrion . ( representative of n = 9 ) ( F - G ) Immunocytochemistry for staining anti ‐ AIF antibodies ( red ) anti ‐ NeuN antibodies ( green ) and nuclei labeled with Hoechst 33342 dye ( blue ) after ( F ) aged SNOC or ( G ) SNOC ( 150 μM ; 4 h ) treatment . Volume rendered 3D reconstruction of confocal image stacks are shown above , and a single confocal plane in grayscale below . Grid , 3 . 3 μm for ( F ) and 1 . 9 μm for ( G ) , scale bars , 5 μm . ( H ) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body , outlined by the NeuN staining ( excluding the nucleus ) . The bar diagram summarizes the mean ± s . e . m . of 282 and 262 neurons measured in n = 19 and 20 image stacks in three independent experiments for aged SNOC and SNOC , respectively ( P ∼ 0 . 99 not significant by Student ' s t ‐ test ) . ( I ) DEVDase activity . Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine , as positive control . The rate of zDEVD ‐ AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units . Data show mean ± s . e . m . for four independent experiments ( * significance at P0 . 01 by ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria. Neurons were co‐transfected with vectors encoding (A) Mito‐DsRed2 and (B) MyrPalm‐mCFP plus (C) AIF‐GFP and the effects of SNOC were recorded using 3D time‐lapse imaging. Projection images zoomed in onto the dendritic arbor of one neuron (representative of n=42). Arrows indicate dendritic spines. The inset shows the soma at appropriate grayscale levels. The apparent brightening of AIF‐GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF. Scale bars, 10 μm. See also Supplementary Video 2.(D) 3D time‐lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition. Scale bar, 10 μm. Five mitochondria were selected and motion tracked throughout the imaging series.(E) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion. Each trace color corresponds to the same colored mitochondrion. (representative of n=9)(F-G) Immunocytochemistry for staining anti‐AIF antibodies (red) anti‐NeuN antibodies (green) and nuclei labeled with Hoechst 33342 dye (blue) after (F) aged SNOC or (G) SNOC (150 μM; 4 h) treatment. Volume rendered 3D reconstruction of confocal image stacks are shown above, and a single confocal plane in grayscale below. Grid, 3.3 μm for (F) and 1.9 μm for (G), scale bars, 5 μm.(H) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body, outlined by the NeuN staining (excluding the nucleus). The bar diagram summarizes the mean±s.e.m. of 282 and 262 neurons measured in n=19 and 20 image stacks in three independent experiments for aged SNOC and SNOC, respectively (P∼0.99 not significant by Student's t‐test).(I) DEVDase activity. Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine, as positive control. The rate of zDEVD‐AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units. Data show mean±s.e.m. for four independent experiments (*significance at P0.01 by ANOVA).


Figure 3 ( A ) Elongated mitochondrion ( arrow ) in neuronal process of the unaffected hemisphere ( top ) . Small mitochondrial segments ( arrows ) are in close proximity to each other in a neuronal process of the ischemic brain region ( bottom ) . Images are representative of three analyzed mice . Scale bars , 500 nm . ( B ) Histogram comparing mitochondrial cross ‐ section length of contralateral , control ( red ) and ischemic ( black ) hemispheres . The lower bins of the histogram ( 2000 nm ) were compared with χ2 ‐ test and were not different . The table shows the presence of long mitochondrial cross ‐ sections ( > 2000 nm ) in the control hemisphere and their absence in the ischemic hemisphere ( P0 . 01 ; Fisher ' s exact test ) . ( C ) Proximities of mitochondria comparing the contralateral ( red ) and ischemic ( black ) hemispheres . Bar diagram shows percents of mitochondrial proximities found to be smaller or larger than 400 nm . The same data set is summarized as a contingency table on the right , ( P0 . 001 ; two ‐ way Fisher ' s exact test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Elongated mitochondrion (arrow) in neuronal process of the unaffected hemisphere (top). Small mitochondrial segments (arrows) are in close proximity to each other in a neuronal process of the ischemic brain region (bottom). Images are representative of three analyzed mice. Scale bars, 500 nm.(B) Histogram comparing mitochondrial cross‐section length of contralateral, control (red) and ischemic (black) hemispheres. The lower bins of the histogram (2000 nm) were compared with χ2‐test and were not different. The table shows the presence of long mitochondrial cross‐sections (>2000 nm) in the control hemisphere and their absence in the ischemic hemisphere (P0.01; Fisher's exact test).(C) Proximities of mitochondria comparing the contralateral (red) and ischemic (black) hemispheres. Bar diagram shows percents of mitochondrial proximities found to be smaller or larger than 400 nm. The same data set is summarized as a contingency table on the right, (P0.001; two‐way Fisher's exact test).


Figure 4Fission is associated with ultrastructural changes in mitochondria . EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission . Cortical cultures were exposed to aged SNOC control ( A - D ) or 200 μM SNOC ( E - L ) for 6 h in the presence of 80 μM zVAD ‐ fmk methyl ester . A slice from each tilt series is shown ( A , E and I ) , followed by orthogonal views of 3D reconstructions after segmentation of the OMM ( blue ) and IMM ( gray ) , with cristae ( various arbitrary colors ) ( B - D , F - H and J - L ) . For clarity , 10 representative cristae are shown in ( B , D , F , G , J and K ) , while all cristae are displayed in ( C , H and L ) . The control mitochondrion ( A - D ) is elongated with a dark matrix , indicating no swelling , and 34 cristae . The NO ‐ exposed mitochondrion in ( E - H ) is dividing , and the lower mitochondrion of the couplet shows severe damage , recognized by an outer membrane rupture , a regionally confined inner membrane blowout , slight matrix swelling and cristae fragmentation . Each of the daughter mitochondria is shorter and rounder than controls . Unlike the upper daughter , few of the cristae extend throughout the lower mitochondrion . Additionally , cristae are considerably smaller and regionally confined . The lower mitochondrion of the couplet contains 82 cristae , while the upper contains only 39 , indicating cristae fragmentation in the former . The NO ‐ exposed mitochondrial triplet in ( I - L ) displays further damage , including membrane degradation , accumulation of an electron dense mass , greater cristae fragmentation , and apparent mitochondrial fragmentation . All cristae are smaller than controls , with 73 in the top mitochondrion , 112 in the middle , and 47 in the bottom . Scale bars , 400 nm . See Supplementary Video 3 , 4 and 5 of tomographic reconstruction .

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Figure 4Fission is associated with ultrastructural changes in mitochondria. EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission. Cortical cultures were exposed to aged SNOC control (A-D) or 200 μM SNOC (E-L) for 6 h in the presence of 80 μM zVAD‐fmk methyl ester. A slice from each tilt series is shown (A, E and I), followed by orthogonal views of 3D reconstructions after segmentation of the OMM (blue) and IMM (gray), with cristae (various arbitrary colors) (B-D, F-H and J-L). For clarity, 10 representative cristae are shown in (B, D, F, G, J and K), while all cristae are displayed in (C, H and L). The control mitochondrion (A-D) is elongated with a dark matrix, indicating no swelling, and 34 cristae. The NO‐exposed mitochondrion in (E-H) is dividing, and the lower mitochondrion of the couplet shows severe damage, recognized by an outer membrane rupture, a regionally confined inner membrane blowout, slight matrix swelling and cristae fragmentation. Each of the daughter mitochondria is shorter and rounder than controls. Unlike the upper daughter, few of the cristae extend throughout the lower mitochondrion. Additionally, cristae are considerably smaller and regionally confined. The lower mitochondrion of the couplet contains 82 cristae, while the upper contains only 39, indicating cristae fragmentation in the former. The NO‐exposed mitochondrial triplet in (I-L) displays further damage, including membrane degradation, accumulation of an electron dense mass, greater cristae fragmentation, and apparent mitochondrial fragmentation. All cristae are smaller than controls, with 73 in the top mitochondrion, 112 in the middle, and 47 in the bottom. Scale bars, 400 nm. See Supplementary Video 3, 4 and 5 of tomographic reconstruction.


Figure 5 ( A ) Projection image of two neurons expressing Mito ‐ DsRed2 ( red ) and MyrPalm ‐ CFP ( blue ) . Images were high ‐ pass filtered and z ‐ stacks were projected over maximum intensity . ( B ) Changes in mitochondrial dynamics were evaluated as mean skeletal length of mitochondria or ( C ) as mitochondrial numbers . ( D ) Magnified representative frames of the time ‐ lapse recording of the area within the white rectangle of ( A ) indicate mitochondrial morphology at time 0 , 20 min and 1 h . These time points are depicted in ( B ) and ( C ) by red arrows . Data are representative of 26 neurons , which exhibited reversible mitochondrial fission from a total of 55 analyzed neurons ( six experiments ) . Scale bar , 10 μm . See Supplementary Video 6 . ( E ) Autophagy in neurites at 6 h after SNOC exposure . Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in the left image . The arrowhead in the right panel indicates membrane wrappers characteristic of autophagosomes . White arrowheads in the insets show the double membranes of the autophagosome and those of mitochondria within the autophagosome . Scale bars , 500 nm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Projection image of two neurons expressing Mito‐DsRed2 (red) and MyrPalm‐CFP (blue). Images were high‐pass filtered and z‐stacks were projected over maximum intensity.(B) Changes in mitochondrial dynamics were evaluated as mean skeletal length of mitochondria or (C) as mitochondrial numbers.(D) Magnified representative frames of the time‐lapse recording of the area within the white rectangle of (A) indicate mitochondrial morphology at time 0, 20 min and 1 h. These time points are depicted in (B) and (C) by red arrows. Data are representative of 26 neurons, which exhibited reversible mitochondrial fission from a total of 55 analyzed neurons (six experiments). Scale bar, 10 μm. See Supplementary Video 6.(E) Autophagy in neurites at 6 h after SNOC exposure. Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in the left image. The arrowhead in the right panel indicates membrane wrappers characteristic of autophagosomes. White arrowheads in the insets show the double membranes of the autophagosome and those of mitochondria within the autophagosome. Scale bars, 500 nm.


Figure 6 ( A ) Mitochondrial fission is linked to a drop in ATP . Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors ( 2 μM rotenone plus 2 μg / ml oligomycin ; Rot / Olig ) . ATP concentrations are shown as the mean ± s . e . m . normalized to the plating density of neurons ( n = 6 ) ( B ) [ ATP ] / [ ADP ] ratios in purified corticalneurons exposed to increasing SNOC concentrations ( n = 4 ) . ( C ) The bar graph depicts ethidine fluorescence density in arbitrary units of aged and fresh SNOC treated neurons at 2 . 5 h . ( D ) Cortical neurons expressing Mito ‐ GFP ( green ) were exposed to aged or fresh 200 μM SNOC solution . After 2 . 5 h cultures were loaded with hydroethidine . Images are representative of more that 20 neurons analyzed ( in D ) for each condition from at least three independent experiments . Scale bar , 10 μm . ( E ) Reduced glutathione ( GSH ) partially blocks mitochondrial fission . Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester , washed once and then exposed to 35 μM SNOC . Mitochondrial fission was scored after 1 h . Data are means ± s . e . m . of quintuplicate samples from one representative experiment of a total of three ( † † † significance at P0 . 001 or n . s . , † not significant compared to control ; * significance at P0 . 05 ) .

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Figure 6(A) Mitochondrial fission is linked to a drop in ATP. Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors (2 μM rotenone plus 2 μg/ml oligomycin; Rot/Olig). ATP concentrations are shown as the mean±s.e.m. normalized to the plating density of neurons (n=6)(B) [ATP]/[ADP] ratios in purified corticalneurons exposed to increasing SNOC concentrations (n=4).(C) The bar graph depicts ethidine fluorescence density in arbitrary units of aged and fresh SNOC treated neurons at 2.5 h.(D) Cortical neurons expressing Mito‐GFP (green) were exposed to aged or fresh 200 μM SNOC solution. After 2.5 h cultures were loaded with hydroethidine. Images are representative of more that 20 neurons analyzed (in D) for each condition from at least three independent experiments. Scale bar, 10 μm.(E) Reduced glutathione (GSH) partially blocks mitochondrial fission. Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester, washed once and then exposed to 35 μM SNOC. Mitochondrial fission was scored after 1 h. Data are means±s.e.m. of quintuplicate samples from one representative experiment of a total of three (†††significance at P0.001 or n.s., †not significant compared to control; *significance at P0.05).


Figure 7 ( A ) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC ( 175 μM ; 7 h ) or rotenone ( 30 nM ; 2 h ) treatments and a comparison of the effects of Aβ25 - 35 versus Aβ35 - 25 ( 10 μM , 6 h ) , as indicated . Scale bar , 20 μm . ( B ) Percentage of mitochondrial fission and cell death at 18 h ( † and † † † significance at P0 . 05 and 0 . 001 as compared to control pcDNA3 transfection ; * , * * and * * * significance at P0 . 05 , 0 . 01 and 0 . 001 , respectively , compared to SNOC treated , pcDNA3 transfected neurons ; n = 3 independent experiments ) . Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining . ( C ) Enforced Drp1 or Fis1 expression ( 60 h ) evokes fission and neuronal cell death ( * , * * and * * * significance at P0 . 05 , 0 . 01 and 0 . 001 , respectively , compared to pcDNA3 transfected neurons ; n = 3 independent experiments ) . ( D ) Rotenone induces dose ‐ dependent mitochondrial fission . The fraction of neurons displaying fissioned mitochondria is shown as the mean ± s . e . m . ( * significance at P0 . 05 , n = 4 ) . ( E ) Mfn1 or Drp1K38A inhibits 100 nM rotenone ‐ induced mitochondrial fission ( 4 h ) and cell death ( 48 h ) . Data indicate means ± s . e . m . of triplicate measurements ( † † and † † † significance at P0 . 01 and 0 . 001 as compared to untreated control ; * * * significance at P0 . 001 as compared to rotenone treatment with pcDNA3 transfection ; n = 3 ) . ( F ) Mitochondrial fission by Aβ35 - 25 or Aβ25 - 35 exposure . The fraction of neurons exhibiting fragmented mitochondria is shown as the mean ± s . e . m . ( † † significance at P0 . 01 compared to pcDNA3 transfected , Aβ35 − 25 treated control ; * and * * significance at P0 . 05 and 0 . 001 , respectively , compared to pcDNA3 transfected , Aβ25 - 35 treated neurons ; n = 3 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC (175 μM; 7 h) or rotenone (30 nM; 2 h) treatments and a comparison of the effects of Aβ25-35 versus Aβ35-25(10 μM, 6 h), as indicated. Scale bar, 20 μm.(B) Percentage of mitochondrial fission and cell death at 18 h († and ††† significance at P0.05 and 0.001 as compared to control pcDNA3 transfection; *,** and *** significance at P0.05 , 0.01 and 0.001, respectively, compared to SNOC treated, pcDNA3 transfected neurons; n=3 independent experiments). Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining.(C) Enforced Drp1 or Fis1 expression (60 h) evokes fission and neuronal cell death (*,** and *** significance at P0.05, 0.01 and 0.001, respectively, compared to pcDNA3 transfected neurons; n=3 independent experiments).(D) Rotenone induces dose‐dependent mitochondrial fission. The fraction of neurons displaying fissioned mitochondria is shown as the mean ±s.e.m. (*significance at P0.05, n=4).(E) Mfn1 or Drp1K38A inhibits 100 nM rotenone‐induced mitochondrial fission (4 h) and cell death (48 h). Data indicate means±s.e.m. of triplicate measurements (†† and ††† significance at P0.01 and 0.001 as compared to untreated control; ***significance at P0.001 as compared to rotenone treatment with pcDNA3 transfection; n=3).(F) Mitochondrial fission by Aβ35-25 or Aβ25-35 exposure. The fraction of neurons exhibiting fragmented mitochondria is shown as the mean±s.e.m. (††significance at P0.01 compared to pcDNA3 transfected, Aβ35−25 treated control; * and ** significance at P0.05 and 0.001, respectively, compared to pcDNA3 transfected, Aβ25-35 treated neurons; n=3).


Figure 3 Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia . Select compounds were injected 24 hours before and on the day of LPS administration , using routes and doses specified in the methods . C57bl / 6 female mice were injected with a high - lethality dose of E . coli LPS ( 38 - 40 μg / g ) followed by a subcutaneous injection of sterile saline . Significant differences in concentration between drug and vehicle - treated pre - and post - pubertal mice are labeled with * * * * ( p < 0 . 0001 ) , * * * ( p < 0 . 001 , * * ( p < 0 . 01 ) , or * ( p < 0 . 05 ) . Percent survival was compared using a log rank Mantel Cox test

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia. Select compounds were injected 24 hours before and on the day of LPS administration, using routes and doses specified in the methods. C57bl/6 female mice were injected with a high-lethality dose of E. coli LPS (38-40 μg/g) followed by a subcutaneous injection of sterile saline. Significant differences in concentration between drug and vehicle-treated pre- and post-pubertal mice are labeled with **** (p<0.0001), *** (p<0.001, ** (p<0.01), or * (p<0.05). Percent survival was compared using a log rank Mantel Cox test


Figure 1A Representative images of alkaline phosphatase ( ALP ) , Alizarin Red S ( ARS ) and Van Gieson ' s staining of human MSCs B , C Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 6 ; * * : p < 0 . 01 and * * * : p < 0 . 001 by t test D , E Quantitative RT - PCR and western blot analyses of the expression of osteogenic markers . Cells were cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by t test F H & E staining of the MSCs - mediated ectopic bone formation . Scale bar , 50 μm G Quantitative analysis of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± SEM ; n = 6 . * : p < 0 . 05 by t

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Representative images of alkaline phosphatase (ALP), Alizarin Red S (ARS) and Van Gieson's staining of human MSCs B, C Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=6; **: p<0.01 and ***: p<0.001 by t test D, E Quantitative RT-PCR and western blot analyses of the expression of osteogenic markers. Cells were cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test F H&E staining of the MSCs-mediated ectopic bone formation. Scale bar, 50 μm G Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=6. *: p<0.05 by t test


Figure 4 A Scatter plot of RNA - seq expression analysis . 508 genes were upregulated and 391 genes were downregulated . Human MSCs were transfected with USP34 or control siRNAs and cultured in osteogenic medium for 2 days . Two biological replicates per group C Heatmap of representative genes associated with BMP2 signaling pathway D Relative activity of BRE luciferase assay . 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours . Results are shown as mean ± SEM ; n = 3 . * * * : p < 0 . 001 by t testE Immunoblot analysis of BMP2 - induce Sp7 expression . Prx1 - Cre ; Usp34f / f and Usp34f / f control MSCs were starved overnight and then stimulated with 100 ng / ml BMP2 for 24 hoursF Quantitative RT - PCR analysis of BMP2 - induce Sp7 transcription . Cells were depleted from serum overnight before BMP2 stimulation . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 and * * : p < 0 . 01 by t testG Representative images of ALP and ARS staining . MSCs were cultured with normal medium supplemented with 100 ng / ml BMP2 for 10 and 21 days , respectivelyH , I Quantitative analyses of the ALP activity ( n = 4 ) and calcium mineralization ( n = 5 ) . Results are shown as mean ± SEM ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by t test J , K Representative microCT images and quantification of newly formed bone in response to BMP2 . Results are shown as mean ± SEM ; n = 8 . * : p < 0 . 05 by t test . Scale bar , 2500 μm L H & E staining of BMP2 - induced newly formed bone . Scale bar , 50 μmM Quantitative analysis of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by t test N H & E staining of calvarial organ cultures stimulated with or without 100 ng / ml BMP2 . Red dotted lines indicate the interface between original and newly formed bone . Scale bar , 50 μmO Quantitative analysis of newly formed bone volume versus original bone volume ( % ) . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 A Scatter plot of RNA-seq expression analysis. 508 genes were upregulated and 391 genes were downregulated. Human MSCs were transfected with USP34 or control siRNAs and cultured in osteogenic medium for 2 days. Two biological replicates per group  C Heatmap of representative genes associated with BMP2 signaling pathway D Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. ***: p<0.001 by t testE Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hoursF Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Cells were depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by t testG Representative images of ALP and ARS staining. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 10 and 21 days, respectivelyH, I Quantitative analyses of the ALP activity (n=4) and calcium mineralization (n=5). Results are shown as mean ± SEM; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test J, K Representative microCT images and quantification of newly formed bone in response to BMP2. Results are shown as mean ± SEM; n=8. *: p<0.05 by t test. Scale bar, 2500 μm L H&E staining of BMP2-induced newly formed bone. Scale bar, 50 μmM Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=8; **: p<0.01 by t test N H&E staining of calvarial organ cultures stimulated with or without 100 ng/ml BMP2. Red dotted lines indicate the interface between original and newly formed bone. Scale bar, 50 μmO Quantitative analysis of newly formed bone volume versus original bone volume (%). Results are shown as mean ± SEM; n=8; **: p<0.01 by t test


Figure 6A Co - immunoprecipitation of USP34 with ectopically expressed FLAG - tagged Smad1 in 293T cells B Immunoblot analysis showing decreased FLAG - Smad1 after USP34 knockdown C Immunoblot of Smad1 - linked polyUb . 293T cells were co - transfected with FLAG - Smad1 overexpression plasmid and USP34 siRNA , and treated with 10 µM MG132 for 4 hours before collection D Immunoblot analysis of the degradation of FLAG - Smad1 protein . 293T cells were co - transfected with FLAG - Smad1 overexpression plasmid and USP34 siRNA , and treated with 50 µg / ml cycloheximide ( CHX ) before collection at designated time points . Right : relative quantification of FLAG - Smad1 protein levels at different time pointsE Co - immunoprecipitation of USP34 with ectopically expressed FLAG - tagged RUNX2 in 293T cellsF Co - immunoprecipitation of USP34 with endogenous RUNX2 in mouse MSCsG Immunoblot of RUNX2 - linked polyUb . Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection . Prx1 - Cre ( + ) indicates MSCs isolated from Prx1 - Cre ; Usp34f / f mice , and Prx1 - Cre ( - ) indicates those from Usp34f / f control miceH Immunoblot analysis of the degradation of endogenous RUNX2 protein in MSCs . Prx1 - Cre ; Usp34f / f and Usp34f / f control MSCs were stimulated with BMP2 , treated with 50 µg / ml CHX , and collected at designated time points . Right : relative quantification of RUNX2 protein levels at different time pointsI Representative images of ALP and ARS staining . Usp34f / f and Prx1 - Cre ; Usp34f / f MSCs were transduced with GFP , Smad1 , Runx2 or Smad1 + Runx2 adenoviruses , and then cultured in osteogenic medium for 5 and 10 days , respectivelyJ , K Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc testL , M H & E staining and quantitative analysis of MSCs - mediated ectopic bone formation . Scale bar , 50 μm . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged Smad1 in 293T cells B Immunoblot analysis showing decreased FLAG-Smad1 after USP34 knockdown  C Immunoblot of Smad1-linked polyUb. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 10 µM MG132 for 4 hours before collection  D Immunoblot analysis of the degradation of FLAG-Smad1 protein. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 50 µg/ml cycloheximide (CHX) before collection at designated time points. Right: relative quantification of FLAG-Smad1 protein levels at different time pointsE Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged RUNX2 in 293T cellsF Co-immunoprecipitation of USP34 with endogenous RUNX2 in mouse MSCsG Immunoblot of RUNX2-linked polyUb. Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection. Prx1-Cre(+) indicates MSCs isolated from Prx1-Cre;Usp34f/f mice, and Prx1-Cre(-) indicates those from Usp34f/f control miceH Immunoblot analysis of the degradation of endogenous RUNX2 protein in MSCs. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were stimulated with BMP2, treated with 50 µg/ml CHX, and collected at designated time points. Right: relative quantification of RUNX2 protein levels at different time pointsI Representative images of ALP and ARS staining. Usp34f/f and Prx1-Cre;Usp34f/f MSCs were transduced with GFP, Smad1, Runx2 or Smad1+Runx2 adenoviruses, and then cultured in osteogenic medium for 5 and 10 days, respectivelyJ, K Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc testL, M H&E staining and quantitative analysis of MSCs-mediated ectopic bone formation. Scale bar, 50 μm. Results are shown as mean ± SEM; n=8; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test


Figure 7A Relative activity of BRE luciferase assay . 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours . Results are shown as mean ± SEM ; n = 3 . * : p < 0 . 05 by ANOVA with Tukey ' s post hoc testB Immunoblot analysis of BMP2 - induce Sp7 expression . Prx1 - Cre ; Usp34f / f MSCs were treated with Smurf1 or control siRNAs , starved overnight and then stimulated with 100 ng / ml BMP2 for 24 hoursC Quantitative RT - PCR analysis of BMP2 - induce Sp7 transcription . Prx1 - Cre ; Usp34f / f MSCs were treated with Smurf1 or control siRNAs , and depleted from serum overnight before BMP2 stimulation . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc testD ALP staining and quantitative measurement of ALP activity . MSCs were cultured with normal medium supplemented with 100 ng / ml BMP2 for 5 days . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc test E Representative images of ARS staining and quantification . Cells were cultured with normal medium supplemented with 100 ng / ml BMP2 for 14 days . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc test F Representative images of ALP and ARS staining . Mouse MSCs were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 and 10 days , respectivelyG , H Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc test I , J Quantitative RT - PC analyses of the expression of osteogenic markers . Cells were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc testwestern blot analyses of the expression of osteogenic markers . Cells were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. *: p<0.05 by ANOVA with Tukey's post hoc testB Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hoursC Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, and depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc testD ALP staining and quantitative measurement of ALP activity. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 5 days. Results are shown as mean ± SEM; n=8; **: p<0.01 by ANOVA with Tukey's post hoc test E Representative images of ARS staining and quantification. Cells were cultured with normal medium supplemented with 100 ng/ml BMP2 for 14 days. Results are shown as mean ± SEM; n=8; *: p<0.05, and **: p<0.01 by ANOVA with Tukey's post hoc test  F Representative images of ALP and ARS staining. Mouse MSCs were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 and 10 days, respectivelyG, H Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test I, J Quantitative RT-PC analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc testwestern blot analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


Figure 1B Phospholipid transport / synthesis , as measured by 3H - serine incorporation , in human fibroblasts ( mean ± SE ; n = 9 , with 5 replicates / experiment ) and in primary mouse hippocampal neurons ( mean ± SE ; n = 3 , with 4 or 5 replicates / experiment ) . * , p < 0 . 05 vs E3 . C Comparison of 3H - serine incorporation in ApoE ACM - treated WT - and Mfn2 - KO MEFs ( mean ± SE ; n = 3 , with 3 replicates / experiment ) . D Duramycin sensitivity in ApoE ACM - treated fibroblasts . Note increased sensitivity after 20 min to 5 μM duramycin in ApoE4 - treated cells , which was blocked upon the addition of 15 μM exogenous PtdEtn ( mean ± SD ; n = 3 , with 3 replicates / experiment ) .

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Figure 1B Phospholipid transport/synthesis, as measured by 3H-serine incorporation, in human fibroblasts (mean±SE; n=9, with 5 replicates/experiment) and in primary mouse hippocampal neurons (mean±SE; n=3, with 4 or 5 replicates/experiment). *, p<0.05 vs E3.C Comparison of 3H-serine incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 3 replicates/experiment).D Duramycin sensitivity in ApoE ACM-treated fibroblasts. Note increased sensitivity after 20 min to 5 μM duramycin in ApoE4-treated cells, which was blocked upon the addition of 15 μM exogenous PtdEtn (mean±SD; n=3, with 3 replicates/experiment).


Figure 2B Fractions from the discontinuous gradient were blotted with anti - ApoE ( WUE - 4 ) antibody ; the doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [ 16 ] . C An ApoE - containing fraction ( fraction 5 [ + ] ) , an ApoE - negative fraction ( fraction 3 [ - ] ) , and recombinant lipid - free ApoE protein were applied to human fibroblasts . Note increase in phospholipid synthesis using an ApoE4 - containing lipoprotein fraction , but not with either an ApoE4 - negative fraction or with lipoprotein - free recombinant ApoE3 or ApoE4 protein ( mean ± SE ; n = 3 , with 3 replicates / experiment ) .

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Figure 2B Fractions from the discontinuous gradient were blotted with anti-ApoE (WUE-4) antibody; the doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [16].C An ApoE-containing fraction (fraction 5 [+]), an ApoE-negative fraction (fraction 3 [-]), and recombinant lipid-free ApoE protein were applied to human fibroblasts. Note increase in phospholipid synthesis using an ApoE4-containing lipoprotein fraction, but not with either an ApoE4-negative fraction or with lipoprotein-free recombinant ApoE3 or ApoE4 protein (mean ± SE; n=3, with 3 replicates/experiment).


Figure 3A Cholesteryl ester synthesis , as measured by 3H - oleate incorporation . Note increased CE in ApoE4 - treated cells , whereas there was essentially no difference in triglyceride ( TAG ) production ( mean ± SE ; n = 3 . with 3 replicates / experiment ) . † , p = 0 . 06 vs E3 . B Comparison of 3H - oleate incorporation in ApoE ACM - treated WT - and Mfn2 - KO MEFs ( mean ± SE ; n = 3 , with 5 replicates / experiment ) . C ApoE ACM - treated fibroblasts were stained with LipidTox green neutral lipid stain , and lipid droplets were visualized ( example at left ) and counted ( right ) . Note significantly more lipid droplets in ApoE4 - treated cells ( mean ± SD , n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Cholesteryl ester synthesis, as measured by 3H-oleate incorporation. Note increased CE in ApoE4-treated cells, whereas there was essentially no difference in triglyceride (TAG) production (mean±SE; n=3. with 3 replicates/experiment). †, p=0.06 vs E3.B Comparison of 3H-oleate incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 5 replicates/experiment).C ApoE ACM-treated fibroblasts were stained with LipidTox green neutral lipid stain, and lipid droplets were visualized (example at left) and counted (right). Note significantly more lipid droplets in ApoE4-treated cells (mean ± SD, n=3).


Figure 4HeLa cells were transfected with plasmids to visualize ER ( in green ) and mitochondria ( in red ) , and then were treated with ApoE3 - and ApoE4 - ACM for 24 hours . Shown is a box - and - whisker plot comparing co - localization in ApoE3 ACM ( n = 15 images analyzed [ gray circles , average of co - localization in ~ 5 cells in each image field ] ) compared to that in ApoE4 ACM ( n = 18 images ) .

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Figure 4HeLa cells were transfected with plasmids to visualize ER (in green) and mitochondria (in red), and then were treated with ApoE3- and ApoE4-ACM for 24 hours. Shown is a box-and-whisker plot comparing co-localization in ApoE3 ACM (n = 15 images analyzed [gray circles, average of co-localization in ~5 cells in each image field]) compared to that in ApoE4 ACM (n = 18 images).


Figure 1A Expression of transcripts coding for PI3K subunit genes in pDCs ( n = 12 biological replicates ) . In the boxplot , the middle , the bottom , and the top of the box represent the median , the 1st quantile and the 3rd quantile , respectively ; the height of the box is given by the difference between the 3rd and the 1st quantiles ( IQR , interquartile range ) ; the whiskers length is 1 . 5 times the interquartile range . Data points beyond the whiskers represent values more extreme than 1 . 5 times the interquartile range above or below the 3rd or the 1st quantile , respectively . C Kyn levels in culture supernatants of pDCs incubated for 24 h with medium alone ( Ctrl ) or IFN - γ after 1 − h pretreatment with PI3K inhibitors . D Real - time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs stimulated for 18 h with TGF - β , in the presence or absence ( Ctrl , control ) of catalytic PI3K inhibitors , normalized to the expression of Gapdh and presented relative to results in untreated cells ( dotted line , 1 - fold ) . E In vivo suppression of the activity of HY - pulsed CD8 − DCs in combination with a minority fraction ( 5 % ) of pDCs conditioned in vitro with TGF - β ( or medium ) for 24 h in the presence or absence ( Ctrl ) of catalytic inhibitors as in C . Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight ( experimental versus control footpads ) . F Real - time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs transfected with p110α - , β - , δ - specific , or negative control ( nc ) siRNA and stimulated for 18 h with TGF - β , normalized to the expression of Gapdh and presented relative to results in unstimulated cells ( dotted line , 1 - fold ) . G In vivo suppression of the activity of HY - pulsed CD8 − DCs in combination with a minority fraction ( 5 % ) of pDCs transfected with siRNA as in ( F ) and conditioned in vitro with TGF - β ( or medium ) for 24 h . Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight ( experimental versus control footpads ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Expression of transcripts coding for PI3K subunit genes in pDCs (n =12 biological replicates). In the boxplot, the middle, the bottom, and the top of the box represent the median, the 1st quantile and the 3rd quantile, respectively; the height of the box is given by the difference between the 3rd and the 1st quantiles (IQR, interquartile range); the whiskers length is 1.5 times the interquartile range. Data points beyond the whiskers represent values more extreme than 1.5 times the interquartile range above or below the 3rd or the 1st quantile, respectively.C Kyn levels in culture supernatants of pDCs incubated for 24 h with medium alone (Ctrl) or IFN-γ after 1−h pretreatment with PI3K inhibitors. D Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs stimulated for 18 h with TGF-β, in the presence or absence (Ctrl, control) of catalytic PI3K inhibitors, normalized to the expression of Gapdh and presented relative to results in untreated cells (dotted line, 1-fold). E In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs conditioned in vitro with TGF-β (or medium) for 24 h in the presence or absence (Ctrl) of catalytic inhibitors as in C. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).F Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs transfected with p110α-, β-, δ-specific, or negative control (nc) siRNA and stimulated for 18 h with TGF-β, normalized to the expression of Gapdh and presented relative to results in unstimulated cells (dotted line, 1-fold). G In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs transfected with siRNA as in (F) and conditioned in vitro with TGF-β (or medium) for 24 h. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).


Figure 2C Pull - down ( PD ) assay of lysates from P1 . HTR cells with unphosphorylated ( YENM ) and tyrosine - phosphorylated IDO1 peptides ( pYENM , pITIM1 , and pITIM2 ) , analyzed by sequential immunoblotting with anti − p85 , − SHP - 1 , and - SHP - 2 antibodies . D Immunoblotting with anti - IDO1 and - p85 antibodies of IDO1 immunoprecipitates obtained from P1 . HTR cells transfected with IDO1 . WT or IDO1 . Y149F . Mock - transfected P1 . HTR cells were used as a control . E Tyrosine phosphatase activity in anti - IDO1 immunoprecipitates from lysates of P1 . HTR transfectants as in D . F Kyn release over 24 h by P1 . HTR transfectants as in D .

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Figure 2C Pull-down (PD) assay of lysates from P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated IDO1 peptides (pYENM, pITIM1, and pITIM2), analyzed by sequential immunoblotting with anti−p85, −SHP-1, and -SHP-2 antibodies.D Immunoblotting with anti-IDO1 and -p85 antibodies of IDO1 immunoprecipitates obtained from P1.HTR cells transfected with IDO1.WT or IDO1.Y149F. Mock-transfected P1.HTR cells were used as a control.E Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from lysates of P1.HTR transfectants as in D.F Kyn release over 24 h by P1.HTR transfectants as in D.


Figure 3A Confocal immunofluorescence microscopy images of P1 . HTR cells either untransfected ( Untr ) or transfected with IDO1 . WT ( WT ) or vector alone ( Mock ) and stained with antibodies recognizing IDO1 ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 20 µm . B Confocal immunofluorescence microscopy images of P1 . HTR cells transfected with IDO1 . WT , co - stained with antibodies recognizing IDO1 ( green ) and markers of intracellular organelles and structures ( as indicated ; red ) . Nuclei were stained with DAPI ( blue ) . The merge imagine is shown for all stainings or without DAPI ( no DAPI ) . Scale bar , 10 µm . C Immunoblotting analysis of EE purified from P1 . IDO1 . WT cells . Mock - transfected cells as in A were used as control . D Distribution of the IDO1 . WT protein expressed by P1 . HTR transfected cells in isopycnic sucrose gradients . Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated .

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Figure 3A Confocal immunofluorescence microscopy images of P1.HTR cells either untransfected (Untr) or transfected with IDO1.WT (WT) or vector alone (Mock) and stained with antibodies recognizing IDO1 (green). Nuclei were stained with DAPI (blue). Scale bar, 20 µm.B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT, co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (as indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.C Immunoblotting analysis of EE purified from P1.IDO1.WT cells. Mock-transfected cells as in A were used as control.D Distribution of the IDO1.WT protein expressed by P1.HTR transfected cells in isopycnic sucrose gradients. Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated.