Figure 1 ( A ) Silver staining of proteins extracted from N2A mouse neuronal cells expressing Flag - HA - tagged C9ORF72 and captured through consecutive anti - Flag and anti - HA affinity purification steps . ( B ) Immunoblot analysis of HA - immunoprecipitated proteins and lysate of HEK293 cells co - expressing HA - tagged C9ORF72 and / or Flag - tagged SMCR8 and / or Flag - tagged WDR41 . ( C ) Coomassie blue staining of Nickel - NTA affinity purification of HIS - C9ORF72 , SMCR8 and WDR41 co - expressed in baculovirus infected insect cells . ( D ) Immunoblot analysis of HA - immunoprecipitated proteins and lysate of HEK293 cells co - expressing HA - tagged C9ORF72 and HA - tagged SMCR8 with various Flag - tagged Rab GTPases . ( E ) Immunoblot against endogenous C9orf72 , Wdr41 , Rab8a , Rab39b , Rab5 and Rab7 of control ( IgG alone ) or endogenous Smcr8 immunoprecipitated from adult mouse brain . ( F ) αP32 - radiolabelled GDP release from GST - tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41 . ( G ) Identical GDP release assay as in ( F ) but using recombinant purified GST - tagged RAB39b instead of RAB8a . Error bars indicate s . e . m . Experiments were repeated 3 times ( 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 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 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 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 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

Figure 1(A) Silver staining of proteins extracted from N2A mouse neuronal cells expressing Flag-HA-tagged C9ORF72 and captured through consecutive anti-Flag and anti-HA affinity purification steps.(B) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and/ or Flag-tagged SMCR8 and/or Flag-tagged WDR41.(C) Coomassie blue staining of Nickel-NTA affinity purification of HIS-C9ORF72, SMCR8 and WDR41 co-expressed in baculovirus infected insect cells.(D) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with various Flag-tagged Rab GTPases.(E) Immunoblot against endogenous C9orf72, Wdr41, Rab8a, Rab39b, Rab5 and Rab7 of control (IgG alone) or endogenous Smcr8 immunoprecipitated from adult mouse brain.(F)αP32-radiolabelled GDP release from GST-tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41.(G) Identical GDP release assay as in (F) but using recombinant purified GST-tagged RAB39b instead of RAB8a. Error bars indicate s.e.m. Experiments were repeated 3 times (n=3).


Figure 2 ( A ) Left panel , representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP - RFP - LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin . Right panel , quantification of LC3B puncta . ( B ) Upper panel , immunoblot analysis of endogenous LC3B ( Map1lc3b ) , C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin . Lower panel , real time RT - qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA . ( C ) Left panel , representative images of immunofluorescence labeling of endogenous P62 ( Sqstm1 ) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , quantification of P62 aggregates . ( D ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP , HA tagged wild type or mutant RAB39b ( CA , Q68L or CN , S22N ) , RAB8a or RAB7 . Right panel , quantification of P62 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . 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 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 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

Figure 2(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP-RFP-LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Right panel, quantification of LC3B puncta.(B) Upper panel, immunoblot analysis of endogenous LC3B (Map1lc3b), C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Lower panel, real time RT-qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA.(C) Left panel, representative images of immunofluorescence labeling of endogenous P62 (Sqstm1) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of P62 aggregates.(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP, HA tagged wild type or mutant RAB39b (CA, Q68L or CN, S22N), RAB8a or RAB7. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


Figure 3 ( A ) Immunoblot analysis of HA - immunoprecipitated proteins and lysate of HEK293 cells co - expressing HA - tagged C9ORF72 and HA - tagged SMCR8 with Flag - tagged TBK1 , NAP1 , TANK or SINTBAD . ( B ) Immunoprecipitated HA - tagged C9ORF72 , HA - tagged SMCR8 and HA - tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32 - radiolabelled ATP . Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography ( upper panel ) , while expression was detected by western blotting ( lower panel ) . ( C ) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS - tagged C9ORF72 : SMCR8 : WDR41 complex purified from baculovirus infected insect cells . ( D ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting the 3 ' UTR of Smcr8 mRNA and plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 ( TA , S402A and T796A ; TD , S402D and T796D ; UD , S400D , S492D , S562D and T666D ) . Right panel , quantification of P62 aggregates . ( E ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 or RAB39b . Right panel , quantification of P62 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . 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 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 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 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 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

Figure 3(A) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with Flag-tagged TBK1, NAP1, TANK or SINTBAD.(B) Immunoprecipitated HA-tagged C9ORF72, HA-tagged SMCR8 and HA-tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32-radiolabelled ATP. Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography (upper panel), while expression was detected by western blotting (lower panel).(C) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS-tagged C9ORF72:SMCR8:WDR41 complex purified from baculovirus infected insect cells.(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting the 3'UTR of Smcr8 mRNA and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 (TA, S402A and T796A; TD, S402D and T796D; UD, S400D, S492D, S562D and T666D). Right panel, quantification of P62 aggregates.(E) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 or RAB39b. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


Figure 4 ( A ) Immunoblot analysis of endogenous Tdp - 43 , C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA . ( B ) Left panel , representative images of immunofluorescence labeling of endogenous phosphorylated Ser409 / 410 - Tdp - 43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , quantification of cytoplasmic Tdp - 43 aggregates . ( C ) Left panel , representative images of immunofluorescence labeling of D169G mutant GFP - tagged TDP - 43 on primary cultures of E18 mouse cortical neurons transfected with either HA - tagged control or HA - C9ORF72 plasmid . Right panel , quantification of cells with cytoplasmic aggregates of TDP - 43 . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * * * indicates p < 0 . 001 . 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 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 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

Figure 4(A) Immunoblot analysis of endogenous Tdp-43, C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA.(B) Left panel, representative images of immunofluorescence labeling of endogenous phosphorylated Ser409/410-Tdp-43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of cytoplasmic Tdp-43 aggregates.(C) Left panel, representative images of immunofluorescence labeling of D169G mutant GFP-tagged TDP-43 on primary cultures of E18 mouse cortical neurons transfected with either HA-tagged control or HA-C9ORF72 plasmid. Right panel, quantification of cells with cytoplasmic aggregates of TDP-43. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


Figure 5 ( A ) Left panel , representative images of organotypic cultures of E18 mouse cortical neurons co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA . Right panel , quantification of ATXN2 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI . ( B ) Cell viability ( tetrazolium assay ) of GT1 - 7 neuronal cells co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA . Error bars indicate s . e . m . Student T - test , * * * indicates p < 0 . 001 . n = 3 . ( C ) Tracing of the swimming trajectories of 48 hours post - fertilization zebrafish larvae following light touch . ( D - F ) Quantification of the touch - evoked swimming distance ( D ) , average velocity ( E ) , and maximum velocity attained ( F ) shows significant functional impairment of the zebrafish injected with HA - tagged ATXN2 with intermediate length of polyQ ( Q30x ) and antisense morpholino oligonucleotides ( AMOs ) against C9orf72 compared to control HA - tagged ATXN2 ( Q22x ) or to the sole injection of AMO against C9orf72 . ( G ) Representative images of motor neurons visualized with anti - Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone . ( H ) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post - fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls . Error bars indicate s . e . m . One - way ANOVA , * * indicates p < 0 . 01 . 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 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 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 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

Figure 5(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA. Right panel, quantification of ATXN2 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI.(B) Cell viability (tetrazolium assay) of GT1-7 neuronal cells co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.(C) Tracing of the swimming trajectories of 48 hours post-fertilization zebrafish larvae following light touch. (D-F) Quantification of the touch-evoked swimming distance (D), average velocity (E), and maximum velocity attained (F) shows significant functional impairment of the zebrafish injected with HA-tagged ATXN2 with intermediate length of polyQ (Q30x) and antisense morpholino oligonucleotides (AMOs) against C9orf72 compared to control HA-tagged ATXN2 (Q22x) or to the sole injection of AMO against C9orf72.(G) Representative images of motor neurons visualized with anti-Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone. (H) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post-fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls. Error bars indicate s.e.m. One-way ANOVA, ** indicates p<0.01. n=3.


Figure 1E and F . TLC analysis of cGAMP , which is indicative of cGAS activation , upon incubation with E ) tRNA or F ) total RNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . The FL - hcGAS concentration was 0 . 265 mg / mL . The tRNA concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , and 0 . 85 mg / mL . The extracted total RNA concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , and 0 . 5 mg / mL . NC indicates the negative control that has only the FL - hcGAS . dsDNA ISD at a concentration of 0 . 05 mg / mL was used as a positive control . All the samples were prepared in 20 mM HEPES at pH 7 . 5 and 150 mM NaCl .

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 O 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 1E and F. TLC analysis of cGAMP, which is indicative of cGAS activation, upon incubation with E) tRNA or F) total RNA (n=2, biological replicates, data from one representative independent biological replicate are shown). The FL-hcGAS concentration was 0.265 mg/mL. The tRNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.85 mg/mL. The extracted total RNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.5 mg/mL. NC indicates the negative control that has only the FL-hcGAS. dsDNA ISD at a concentration of 0.05 mg/mL was used as a positive control. All the samples were prepared in 20 mM HEPES at pH 7.5 and 150 mM NaCl.


Figure 2F . Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments . G . The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells ( n = 13 , biological replicates , data from one representative independent biological replicate are shown ) . The scale bar represents 10 µm . The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area ( yellow circle ) . H . The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells ( n = 9 , biological replicates , data from one representative independent biological replicate are shown ) . The scale bar represents 10 µm . The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area ( yellow circle ) . I . Pearson ' s correlation coefficient ( CC ) analysis of the fluorescence signals of cGAS vs . RNA ( n = 13 , biological replicates ) , and mCherry vs . RNA ( n = 9 , biological replicates ) . The median values were calculated and used for evaluation of significance . * * * P < 0 . 001 ( two - tailed Mann - Whitney test ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2F. Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments.G. The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells (n=13, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). H. The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells (n=9, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). I. Pearson's correlation coefficient (CC) analysis of the fluorescence signals of cGAS vs. RNA (n=13, biological replicates), and mCherry vs. RNA (n=9, biological replicates). The median values were calculated and used for evaluation of significance. ***P < 0.001 (two-tailed Mann-Whitney test).


Figure 4D . Fluorescence microscopy images showing the replacement of FTSC - tRNA by Cy5 - ISD at indicated concentrations in preformed granules of hcGAS and FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative area that is 1 / 16 of the raw image is displayed . The scale bars represent 20 µm . E . Fluorescence microscopy images showing the replacement of FTSC - tRNA by TAMRA - 380 bp - dsDNA at indicated concentrations in preformed granules of hcGAS and FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative area that is 1 / 16 of the raw image is displayed . The scale bars represent 20 µm . F . Quantification of the replacement in " D " . For each square , the raw image used was evenly divided into four parts . Fluorescence signal ratios of Cy5 - ISD over FTSC - tRNA in the four parts were calculated , averaged and plotted . G . Quantification of the replacement in " E " . For each square , the raw image used was evenly divided into four parts . Fluorescence signal ratios of TAMRA - 380 bp - dsDNA over FTSC - tRNA in the four parts were calculated , averaged and plotted .

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Figure 4D. Fluorescence microscopy images showing the replacement of FTSC-tRNA by Cy5-ISD at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. E. Fluorescence microscopy images showing the replacement of FTSC-tRNA by TAMRA-380 bp-dsDNA at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. F. Quantification of the replacement in "D". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of Cy5-ISD over FTSC-tRNA in the four parts were calculated, averaged and plotted. G. Quantification of the replacement in "E". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of TAMRA-380 bp-dsDNA over FTSC-tRNA in the four parts were calculated, averaged and plotted.


Figure 1 The 3 ' - UTR sequence of mUcp1 RNA . Canonical CPEs ( UUUUA1 - 2U in bold ) , the reported BRF1 - binding site ( UAUUUAU , gray box ) and predicted IMP2 - binding motifs ( AYAYA and YA2YA , underlined ) are denoted . Ucp1L and Ucp1S carrying long and short 3 ' - UTRs , respectively , are resulted from alternative use of two polyadenylation signals ( Hex : AAUAAA , open box ) . ORF , open reading frame . The arrowhead marks the end of Ucp1S sequence . Northern blots of Ucp1 using 5 µg BAT total RNA and the denoted radiolabeled probes Schematic comparison of mUcp1L and hUcp1 . Poly ( A ) signal ( Hex , open box ) and CPE ( black box ) are denoted . The RT - PCR results from human , WT ( Ucp1 + / + ) and UCP1 - KO ( Ucp1 - / - ) BAT RNA samples Dual luciferase reporter assay and RT - qPCR . The reporter plasmids , Fluc - mUcp1S , - mUcp1L or - hUcp1L 3 ' - UTR , and Rluc were co - transfected into differentiated HIB1B adipocytes . The transfected cells were used for luciferase activity assay and RT - qPCR . Data are mean ± SD from 3 independent experiments . Translation efficiency was defined as normalized activity ( Fluc / Rluc ) divided by normalized RNA level ( Fluc / Rluc ) . * * * p < 0 . 001 compared with Fluc - mUcp1S , one - way ANOVA

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Figure 1 The 3'-UTR sequence of mUcp1 RNA. Canonical CPEs (UUUUA1-2U in bold), the reported BRF1-binding site (UAUUUAU, gray box) and predicted IMP2-binding motifs (AYAYA and YA2YA, underlined) are denoted. Ucp1L and Ucp1S carrying long and short 3'-UTRs, respectively, are resulted from alternative use of two polyadenylation signals (Hex: AAUAAA, open box). ORF, open reading frame. The arrowhead marks the end of Ucp1S sequence. Northern blots of Ucp1 using 5 µg BAT total RNA and the denoted radiolabeled probes  Schematic comparison of mUcp1L and hUcp1. Poly(A) signal (Hex, open box) and CPE (black box) are denoted. The RT-PCR results from human, WT (Ucp1+/+) and UCP1-KO (Ucp1-/-) BAT RNA samples  Dual luciferase reporter assay and RT-qPCR. The reporter plasmids, Fluc-mUcp1S, -mUcp1L or -hUcp1L 3'-UTR, and Rluc were co-transfected into differentiated HIB1B adipocytes. The transfected cells were used for luciferase activity assay and RT-qPCR. Data are mean ± SD from 3 independent experiments. Translation efficiency was defined as normalized activity (Fluc/Rluc) divided by normalized RNA level (Fluc/Rluc). ***p < 0.001 compared with Fluc-mUcp1S, one-way ANOVA


Figure 2 Northern blotting of Ucp1 and 18S rRNA and RT - PCR of Ucp1L and Ucp1S using total RNAs from the denoted BAT samples ( 2 mice per genotype , 2 - 3 mo old ) . The short PCR fragments were isolated for sequencing Western blot analysis using WT and Ucp1 ∆ L BAT lysates . The protein level of UCP1 was normalized to GAPDH ( 4 mo old , n = 4 per group ) . * * p < 0 . 01 , Student ' s t test Dual luciferase reporter assay . The reporter plasmids , Fluc - mUcp1L 3 ' - UTR , and Rluc were co - transfected with the plasmid expressing EGFP , or myc - tagged CPEB ( immunoblots shown at the left ) into differentiated HIB1B adipocytes ( Data from 3 independent experiments ) . * p < 0 . 05 compared with EGFP , one - way ANOVA Western blot analysis using BAT lysates prepared from various CPEB - WT and KO male littermates ( 4 mo old , n = 4 per group ) . * p < 0 . 05 , Student ' s t test

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Figure 2 Northern blotting of Ucp1 and 18S rRNA and RT-PCR of Ucp1L and Ucp1S using total RNAs from the denoted BAT samples (2 mice per genotype, 2-3 mo old). The short PCR fragments were isolated for sequencing  Western blot analysis using WT and Ucp1∆L BAT lysates. The protein level of UCP1 was normalized to GAPDH (4 mo old, n = 4 per group). **p < 0.01, Student's t test  Dual luciferase reporter assay. The reporter plasmids, Fluc-mUcp1L 3'-UTR, and Rluc were co-transfected with the plasmid expressing EGFP, or myc-tagged CPEB (immunoblots shown at the left) into differentiated HIB1B adipocytes (Data from 3 independent experiments). *p < 0.05 compared with EGFP, one-way ANOVA  Western blot analysis using BAT lysates prepared from various CPEB-WT and KO male littermates (4 mo old, n = 4 per group). *p < 0.05, Student's t test


Figure 3 Northern blotting of Ucp1L and Ucp1S in BAT from CPEB2 - WT and - KO male mice at room temperature or after 5 - h cold exposure ( CE ) at 4oC ( 5 mice per genotype , 4 - 7 mo old )

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Figure 3 Northern blotting of Ucp1L and Ucp1S in BAT from CPEB2-WT and -KO male mice at room temperature or after 5-h cold exposure (CE) at 4oC (5 mice per genotype, 4-7 mo old)


Figure 4Western blot analysis and RT - qPCR analysis of protein and mRNA levels of UCP1 and GAPDH in WT and KO male BAT ( 13 mo old , n = 3 - 4 per group ) Western blot analysis of UCP1 and GAPDH in BAT lysates from WT and KO female mice at 4 and 12 mo old ( n = 3 per group ) . Protein levels were normalized to that of GAPDH . RT - qPCR analysis of Ucp1 mRNA level in BAT collected from ( B ) relative to Gapdh mRNA levelHematoxylin and eosin sections of BAT from WT and CPEB2 - KO female mice at 4 and 12 mo old . Scale bars , 1 mmLipid droplet size and cell number ( i . e . , number of nuclei ) in WT and KO BAT ( representative images shown in 4D ) were quantified and displayed as arbitrary units . Three tissue sections from each BAT and 3 mice per group were analyzedWeight and representative images of WT and KO BAT . Three mice per group were

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Figure 4Western blot analysis and RT-qPCR analysis of protein and mRNA levels of UCP1 and GAPDH in WT and KO male BAT (13 mo old, n = 3-4 per group)Western blot analysis of UCP1 and GAPDH in BAT lysates from WT and KO female mice at 4 and 12 mo old (n = 3 per group). Protein levels were normalized to that of GAPDH. RT-qPCR analysis of Ucp1 mRNA level in BAT collected from (B) relative to Gapdh mRNA levelHematoxylin and eosin sections of BAT from WT and CPEB2-KO female mice at 4 and 12 mo old. Scale bars, 1 mmLipid droplet size and cell number (i.e., number of nuclei) in WT and KO BAT (representative images shown in 4D) were quantified and displayed as arbitrary units. Three tissue sections from each BAT and 3 mice per group were analyzedWeight and representative images of WT and KO BAT. Three mice per group were analyzed


Figure 6 CPEB2 - WT and - KO mice were transduced with adeno - associated virus ( AAV ) expressing full - length ( AAV _ CP2 ) or RNA - binding domain ( AAV _ RBD ) of CPEB2 . Immunostaining of CPEB2 and UCP1 in KO BAT transduced with AAV _ CP2 for 2 weeks , with Hoechst staining of nuclei . Scale bar , 10 µm . See more images in Fig EV4D Western blot analysis of UCP1 in BAT lysates from WT and KO male mice 2 weeks after AAV transduction ( n = 6 mice per group ) . * p < 0 . 05 , Student ' s t test BAT temperature of WT and KO male mice after 2 weeks of AAV transduction measured during 12 - h light time ( n = 7 mice per group ) . * p < 0 . 05 , Student ' s t test BAT temperature in WT and KO male mice , and of AAV _ CP2 - transduced WT and KO male mice ( n = 7 mice per group ) before and after i . p . injection of CL316243 ( CL ) . * p < 0 . 05 , repeated - measures two - way ANOVA

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Figure 6 CPEB2-WT and -KO mice were transduced with adeno-associated virus (AAV) expressing full-length (AAV_CP2) or RNA-binding domain (AAV_RBD) of CPEB2. Immunostaining of CPEB2 and UCP1 in KO BAT transduced with AAV_CP2 for 2 weeks, with Hoechst staining of nuclei. Scale bar, 10 µm. See more images in Fig EV4D  Western blot analysis of UCP1 in BAT lysates from WT and KO male mice 2 weeks after AAV transduction (n = 6 mice per group). *p < 0.05, Student's t test  BAT temperature of WT and KO male mice after 2 weeks of AAV transduction measured during 12-h light time (n = 7 mice per group). *p < 0.05, Student's t test  BAT temperature in WT and KO male mice, and of AAV_CP2-transduced WT and KO male mice (n = 7 mice per group) before and after i.p. injection of CL316243 (CL). * p < 0.05, repeated-measures two-way ANOVA


Figure 2Normalized RPF count profile averaged for all E . coli transcripts . Profiles generated for cells grown in the absence and presence of IPTG ( 1 mM ) . Start and stop codons are shaded . Bar chart of all measured RBS initiation rates ranked by their strength . Strong RBSs with initiation rates > 1 ribosome / s are highlighted in red . ( D ) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength . Stop codons with translation termination efficiency > 0 . 99 are highlighted in red . ( E ) Distribution of initiation rates for cells grown in the absence and presence of IPTG ( 1 mM ) . ( F ) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG ( 1 mM ) .

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Figure 2Normalized RPF count profile averaged for all E. coli transcripts. Profiles generated for cells grown in the absence and presence of IPTG (1 mM). Start and stop codons are shaded.Bar chart of all measured RBS initiation rates ranked by their strength. Strong RBSs with initiation rates >1 ribosome/s are highlighted in red. (D) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength. Stop codons with translation termination efficiency >0.99 are highlighted in red. (E) Distribution of initiation rates for cells grown in the absence and presence of IPTG (1 mM). (F) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG (1 mM).


Figure 3Comparison of protein synthesis rate of endogenous E . coli genes measured using Ribo - seq from this study ( in molecules / s units ) and from that by Li et al . ( Li et al , 2014 ) ( in molecules / generation units ) . Each point corresponds to a single gene and color denotes the ratio of transcription initiation rate to translation initiation rate ( giving RNAP / ribosome ) capturing whether transcription ( light yellow ) or translation ( dark blue ) is more dominant . Transcription ( bottom ) and translation ( top ) profiles for yggN , rpoH and greA , computed from the RNA - seq and Ribo - seq data without induction . Positions of the genetic parts and gene are shown below the profiles . ( C ) Promoter strengths in RNAP / s units and RBS initiation rates in ribosome / s units . Transcription ( bottom ) and translation ( top ) profiles for lacZ . Profiles are shown for cells in the absence and presence of IPTG ( 1 mM ) . Position of genetic parts and gene is shown below the profiles . RBS is omitted from the genetic design due to its size . ( E ) Measured promoter strength in RNAP / s units , RBS initiation rate in ribosomes / s units , and the transcriptional terminator and translation termination efficiency for lacZ . Data shown for cells in the absence and presence of IPTG ( 1 mM ) .

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Figure 3Comparison of protein synthesis rate of endogenous E. coli genes measured using Ribo-seq from this study (in molecules/s units) and from that by Li et al. (Li et al, 2014) (in molecules/generation units). Each point corresponds to a single gene and color denotes the ratio of transcription initiation rate to translation initiation rate (giving RNAP/ribosome) capturing whether transcription (light yellow) or translation (dark blue) is more dominant.Transcription (bottom) and translation (top) profiles for yggN, rpoH and greA, computed from the RNA-seq and Ribo-seq data without induction. Positions of the genetic parts and gene are shown below the profiles. (C) Promoter strengths in RNAP/s units and RBS initiation rates in ribosome/s units.Transcription (bottom) and translation (top) profiles for lacZ. Profiles are shown for cells in the absence and presence of IPTG (1 mM). Position of genetic parts and gene is shown below the profiles. RBS is omitted from the genetic design due to its size. (E) Measured promoter strength in RNAP/s units, RBS initiation rate in ribosomes/s units, and the transcriptional terminator and translation termination efficiency for lacZ. Data shown for cells in the absence and presence of IPTG (1 mM).


Figure 4Translation profiles for the PK - LacZ construct in cells cultured in the absence ( bottom ) and presence ( top ) of IPTG ( 1 mM ) . The gene10 , middle , and lacZ regions are labelled above the profiles . Shaded region denotes the PK , and dashed lines denote the start codon and stop codons of gene10 and LacZ . Fraction of the total RPFs and mRNA reads in each reading frame for the gene10 , PK or middle , and lacZ regions schematically shown below and are of the PK - LacZ construct . Data shown separately for cells cultured in the absence and presence of IPTG ( 1 mM ) . Violin plots of the distributions of fractions of total RPFs and mRNA reads in each reading frame for all E . coli transcripts .

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Figure 4Translation profiles for the PK-LacZ construct in cells cultured in the absence (bottom) and presence (top) of IPTG (1 mM). The gene10, middle, and lacZ regions are labelled above the profiles. Shaded region denotes the PK, and dashed lines denote the start codon and stop codons of gene10 and LacZ.Fraction of the total RPFs and mRNA reads in each reading frame for the gene10, PK or middle, and lacZ regions schematically shown below and are of the PK-LacZ construct. Data shown separately for cells cultured in the absence and presence of IPTG (1 mM).Violin plots of the distributions of fractions of total RPFs and mRNA reads in each reading frame for all E. coli transcripts.


Figure 5Change in expression of chromosomal genes in E . coli cells following induction of PK - lacZ expression ( 1 mM IPTG ) . Each point represents a transcript . Differentially expressed genes ( mRNA count : P < 0 . 001 and absolute log2 fold - change > 1 . 37 ; translation efficiency : P < 0 . 01 ) are highlighted in color and by an alternative point shape ( transcriptional regulation : purple cross ; translational regulation : orange open circle ) . Translation initiation rates for all E . coli RBSs in cells harboring the LacZ and PK - LacZ constructs in the absence and presence of IPTG ( 1 mM ) . Solid line shows the same initiation rate for both conditions . Dotted lines denote linear regressions for the data with no offset . Fractions of mRNA reads and RPFs mapping to each synthetic expression construct ( LacZ and PK - LacZ ) and E . coli transcripts , which are divided into three major categories : ribosomal , metabolic , and other functions . Data shown for cells cultured in the absence and presence of IPTG ( 1 mM ) .

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Figure 5Change in expression of chromosomal genes in E. coli cells following induction of PK-lacZ expression (1 mM IPTG). Each point represents a transcript. Differentially expressed genes (mRNA count: P < 0.001 and absolute log2 fold-change > 1.37; translation efficiency: P < 0.01) are highlighted in color and by an alternative point shape (transcriptional regulation: purple cross; translational regulation: orange open circle).Translation initiation rates for all E. coli RBSs in cells harboring the LacZ and PK-LacZ constructs in the absence and presence of IPTG (1 mM). Solid line shows the same initiation rate for both conditions. Dotted lines denote linear regressions for the data with no offset.Fractions of mRNA reads and RPFs mapping to each synthetic expression construct (LacZ and PK-LacZ) and E. coli transcripts, which are divided into three major categories: ribosomal, metabolic, and other functions. Data shown for cells cultured in the absence and presence of IPTG (1 mM).


Figure 1B HeLa cells were infected with x - light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , and then samples were labelled with antibody for SEPT7 . The scale bar represents 5 μm . Graph represents mean % ± SEM of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 4 independent experiments . Student ' s t - test , * * * = p < 0 . 001 . C HeLa cells were infected with x - light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , and then samples were labelled with antibody for p62 . The scale bar represents 5 μm . Graph represents mean % ± SEM of Shigella responding to IPTG without ( - ) or with ( + ) p62 from at least 4 independent experiments . Student ' s t - test , * * * = p < 0 . 001 . D Control ( CTRL ) or isopropanol - treated x - light Shigella GFP were induced with IPTG for 30 min , then labelled with SYTOX Orange for 10 min . The scale bar represents 5 μm . E HeLa cells were infected for 1 h 30 min , treated with ethanol ( CTRL ) or erythromycin ( EM ) for 2 h prior to fixation , and then labelled with antibody for SEPT7 . Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments . Student ' s t - test , * * * = p < 0 . 001 .

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Figure 1B HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 4 independent experiments. Student's t-test, *** = p<0.001.C HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for p62. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG without (-) or with (+) p62 from at least 4 independent experiments. Student's t-test, *** = p<0.001.D Control (CTRL) or isopropanol-treated x-light Shigella GFP were induced with IPTG for 30 min, then labelled with SYTOX Orange for 10 min. The scale bar represents 5 μm.E HeLa cells were infected for 1 h 30 min, treated with ethanol (CTRL) or erythromycin (EM) for 2 h prior to fixation, and then labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.


Figure 2B HeLa cells stably expressing SEPT6 - GFP were infected with S . flexneri AfaI for 4 h , harvested , and then tested with Co - IP . Empty vector and / or uninfected HeLa cells were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from cells , and extracts were immunoblotted for SEPT6 , GFP , SEPT7 , or p62 . The lysates were immunoblotted for GAPDH as control for cellular protein levels .

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Figure 2B HeLa cells stably expressing SEPT6-GFP were infected with S. flexneri AfaI for 4 h, harvested, and then tested with Co-IP. Empty vector and/or uninfected HeLa cells were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for SEPT6, GFP, SEPT7, or p62. The lysates were immunoblotted for GAPDH as control for cellular protein levels.


Figure 3A HeLa cells were labelled with MitoTracker Red CMXRos , treated with cytochalasin D for 30 min , then fixed and labelled for endogenous SEPT7 for confocal microscopy . The scale bar represents 0 . 5 μm . See also Fig EV3A . B HeLa cells were infected with S . flexneri for 4 h 40 min , labelled with MitoTracker Red CMXRos , fixed and labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy . The scale bar represents 1 μm . C HeLa cells stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , infected with Shigella - mCherry for 4 h 40 min , and processed for CLEM . SEPT6 is shown in green , mitochondria in red , and Shigella - mCherry in blue . Septin cages identified by fluorescent light microscopy ( FLM ) were processed for TEM . The right - most image is enlarged from the boxed region in the TEM image , and shows the bacterium ( Sf ) surrounded by a double membrane of autophagy ( Au ) and the mitochondrial membrane ( Mt ) . The scale bar represents 1 μm . See also Fig EV3B . D HeLa cells stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , then infected with Shigella - mCherry for 2 h for live confocal microscopy . Time frame sequence shows a septin cage assembling around Shigella . Mitochondria ( circle ) support sites of septin ring assembly around the bacterium . Each frame was acquired every 2 min . The scale bar represents 1 μm . See also Movie EV1 . E HeLa cells were treated with control ( CTRL ) , Drp1 , or Mfn1 siRNA for 72 h . Whole cell lysates were immunoblotted for Drp1 or Mfn1 to show the efficiency of depletion . GAPDH was used as loading control . siRNA - treated cells were infected with S . flexneri for 4 h 40 min , then fixed for microscopy and stained for endogenous SEPT7 for quantitative confocal microscopy . Graph represents the mean % ± SEM of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment . Student ' s t - test , * * = p < 0 . 01 ; * * * = p < 0 . 001 .

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Figure 3A HeLa cells were labelled with MitoTracker Red CMXRos, treated with cytochalasin D for 30 min, then fixed and labelled for endogenous SEPT7 for confocal microscopy. The scale bar represents 0.5 μm. See also Fig EV3A.B HeLa cells were infected with S. flexneri for 4 h 40 min, labelled with MitoTracker Red CMXRos, fixed and labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy. The scale bar represents 1 μm.C HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, infected with Shigella-mCherry for 4 h 40 min, and processed for CLEM. SEPT6 is shown in green, mitochondria in red, and Shigella-mCherry in blue. Septin cages identified by fluorescent light microscopy (FLM) were processed for TEM. The right-most image is enlarged from the boxed region in the TEM image, and shows the bacterium (Sf) surrounded by a double membrane of autophagy (Au) and the mitochondrial membrane (Mt). The scale bar represents 1 μm. See also Fig EV3B.D HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, then infected with Shigella-mCherry for 2 h for live confocal microscopy. Time frame sequence shows a septin cage assembling around Shigella. Mitochondria (circle) support sites of septin ring assembly around the bacterium. Each frame was acquired every 2 min. The scale bar represents 1 μm. See also Movie EV1.E HeLa cells were treated with control (CTRL), Drp1, or Mfn1 siRNA for 72 h. Whole cell lysates were immunoblotted for Drp1 or Mfn1 to show the efficiency of depletion. GAPDH was used as loading control. siRNA-treated cells were infected with S. flexneri for 4 h 40 min, then fixed for microscopy and stained for endogenous SEPT7 for quantitative confocal microscopy. Graph represents the mean % ± SEM of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment. Student's t-test, ** = p <0.01; *** = p<0.001.


Figure 4A HeLa cells expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos , and fixed for confocal microscopy . Inset image highlights location of septins at sites of mitochondrial fission . The scale bar represents 5 μm . B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP , and processed for TEM . The right image is enlarged from the boxed region in the TEM image , and shows a SEPT7 ( S7 ) filament ( labelled in black ) intersecting with the mitochondrial ( Mt ) fission site . The scale bar represents 100 nm . See also Fig EV4 . C HeLa cells expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos for live SOFI - based super resolution microscopy . Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI - based algorithm . Arrows highlight SEPT6 association with location of mitochondrial fission . The scale bar represents 1 µm . See also Movie EV3 . D HeLa cells expressing SEPT6 - GFP were labelled with MitoTracker Red CMXRos , fixed for widefield immunofluorescence microscopy , and labelled with antibodies to Drp1 . Arrows highlight one example of Drp1 and SEPT6 association with location of mitochondrial fission . The scale bar represents 1 μm . E HeLa cells were transfected with Mito - BFP and ActA - SEPT7 - mCherry , fixed and labelled with antibody to Drp1 for widefield immunofluorescence microscopy . The scale bar represents 5 μm . Inset images highlight Drp1 and SEPT7 associated with sites of mitochondrial fission . Pearson ' s correlation coefficient from 3 independent experiments ( mean ± SEM ) shows that mitochondria in ActA - SEPT7 transfected cells recruit significantly more Drp1 than control cells ( Mito - BFP transfected alone ) . Student ' s t - test , * = p < 0 . 05 .

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Figure 4A HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos, and fixed for confocal microscopy. Inset image highlights location of septins at sites of mitochondrial fission. The scale bar represents 5 μm.B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP, and processed for TEM. The right image is enlarged from the boxed region in the TEM image, and shows a SEPT7 (S7) filament (labelled in black) intersecting with the mitochondrial (Mt) fission site. The scale bar represents 100 nm. See also Fig EV4.C HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos for live SOFI-based super resolution microscopy. Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI-based algorithm. Arrows highlight SEPT6 association with location of mitochondrial fission. The scale bar represents 1 µm. See also Movie EV3.D HeLa cells expressing SEPT6-GFP were labelled with MitoTracker Red CMXRos, fixed for widefield immunofluorescence microscopy, and labelled with antibodies to Drp1. Arrows highlight one example of Drp1 and SEPT6 association with location of mitochondrial fission. The scale bar represents 1 μm.E HeLa cells were transfected with Mito-BFP and ActA-SEPT7-mCherry, fixed and labelled with antibody to Drp1 for widefield immunofluorescence microscopy. The scale bar represents 5 μm. Inset images highlight Drp1 and SEPT7 associated with sites of mitochondrial fission. Pearson's correlation coefficient from 3 independent experiments (mean ± SEM) shows that mitochondria in ActA-SEPT7 transfected cells recruit significantly more Drp1 than control cells (Mito-BFP transfected alone). Student's t-test, * = p<0.05.


Figure 5A HeLa cells were treated with control ( CTRL ) , SEPT7 , or Drp1 siRNA for 72 h . Whole cell lysate of siRNA - treated cells were immunoblotted for GAPDH , SEPT7 , or Drp1 to show the efficiency of siRNA depletion . GAPDH was used as loading control . siRNA - treated cells were labelled with MitoTracker Red CMXRos , and fixed for confocal microscopy . The scale bar represents 5 μm . B HeLa cells were treated with control ( CTRL ) , SEPT2 , SEPT7 , SEPT9 , Drp1 , or SEPT7 + Drp1 siRNA for 72 h , labelled with MitoTracker Red CMXRos , and fixed for quantitative confocal microscopy . Graph represents the distribution in length ( μm ; whiskers from min to max ) of mitochondria in siRNA - treated cells from 3 independent experiments ( analysis of at least 100 measurements per biological replicate ) . Student ' s t - test , * * * = p < 0 . 001 . C HeLa cells stably expressing SEPT6 - GFP were harvested , and then tested with Co - IP . HeLa cells expressing GFP were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from cells , and extracts were immunoblotted for Drp1 , SEPT2 , SEPT7 , or GFP . The lysates were immunoblotted for GAPDH as control for cellular protein levels . D HeLa cells were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , and whole cell lysates were immunoblotted for SEPT7 , Drp1 , phosphorylated Drp1 ( P - Drp1 ) , or Mfn1 . GAPDH was used as loading control . Graph represents the mean % ± SEM of the relative amount of Drp1 , P - Drp1 , Mfn1 , or SEPT7 proteins quantified by densitometry from 4 independent experiments . Student ' s t - test , ns = non - significant ; * * = p < 0 . 01 , * * * = p < 0 . 001 . E HeLa cells were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed and labelled for endogenous P - Drp1 for quantitative confocal microscopy . Representative images shown here . The scale bar represents 1 μm . The recruitment of P - Drp1 to mitochondria was significantly decreased as compared to control cells as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± SEM , analysis of at least 250 cells per biological replicate ) . Student ' s t - test , * = p < 0 . 05 . F HeLa cells were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed and labelled for endogenous Mfn1 for quantitative confocal microscopy . Representative images shown here . The scale bar represents 1 μm . The recruitment of Mfn1 to mitochondria was significantly increased as compared to control cells as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± SEM , analysis of at least 250 cells per biological replicate ) . Student ' s t - test , * = p < 0 . 05 .

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Figure 5A HeLa cells were treated with control (CTRL), SEPT7, or Drp1 siRNA for 72 h. Whole cell lysate of siRNA-treated cells were immunoblotted for GAPDH, SEPT7, or Drp1 to show the efficiency of siRNA depletion. GAPDH was used as loading control. siRNA-treated cells were labelled with MitoTracker Red CMXRos, and fixed for confocal microscopy. The scale bar represents 5 μm.B HeLa cells were treated with control (CTRL), SEPT2, SEPT7, SEPT9, Drp1, or SEPT7+ Drp1 siRNA for 72 h, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Graph represents the distribution in length (μm; whiskers from min to max) of mitochondria in siRNA-treated cells from 3 independent experiments (analysis of at least 100 measurements per biological replicate). Student's t-test, *** = p<0.001.C HeLa cells stably expressing SEPT6-GFP were harvested, and then tested with Co-IP. HeLa cells expressing GFP were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for Drp1, SEPT2, SEPT7, or GFP. The lysates were immunoblotted for GAPDH as control for cellular protein levels.D HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, and whole cell lysates were immunoblotted for SEPT7, Drp1, phosphorylated Drp1 (P-Drp1), or Mfn1. GAPDH was used as loading control. Graph represents the mean % ± SEM of the relative amount of Drp1, P-Drp1, Mfn1, or SEPT7 proteins quantified by densitometry from 4 independent experiments. Student's t-test, ns = non-significant; ** = p<0.01, *** = p<0.001.E HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous P-Drp1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of P-Drp1 to mitochondria was significantly decreased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.F HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous Mfn1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of Mfn1 to mitochondria was significantly increased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.


Figure 6A HeLa cells stably expressing SEPT6 - GFP were treated with Drp1 siRNA for 72 h , transfected with mito - BFP 24 h , and then infected with S . flexneri - mCherry for 2 h for live confocal microscopy . Representative image shows interplay between two Shigella - septin cages and fused mitochondria . The scale bar represents 1 μm . See also Movie EV3 . B HeLa cells were treated with Drp1 siRNA for 72 h , and then infected with x - light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , and then samples were labelled with antibody for SEPT7 . Graph represents mean % ± SEM of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments . Student ' s t - test , * * * = p < 0 . 001 . C HeLa cells were infected with S . flexneri M90T for 4 h 40 min , labelled with MitoTracker Red CMXRos , and fixed for quantitative confocal microscopy . Boxplots show the length of mitochondrial ( μm ; whiskers from min to max ) surrounding Shigella outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments ( analysis of at least 130 measurements per biological replicate ) . Student ' s t - test ; * * * = p < 0 . 001 . D HeLa cells were either kept uninfected as a control or infected with S . flexneri M90T or S . flexneri ΔIcsA for 4 h 40 min . Samples were labelled with MitoTracker Red CMXRos , and fixed for quantitative confocal microscopy . Boxplots show length of mitochondria ( μm ; whiskers from min to max ) in uninfected cells ( CTRL ) , surrounding S . flexneri M90T ( + M90T ) or S . flexneri ΔIcsA ( + ΔIcsA ) from 3 independent experiments ( analysis of at least 250 measurements per biological replicate ) . Student ' s t - test ; * * * = p < 0 . 001 .

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Figure 6A HeLa cells stably expressing SEPT6-GFP were treated with Drp1 siRNA for 72 h, transfected with mito-BFP 24 h, and then infected with S. flexneri-mCherry for 2 h for live confocal microscopy. Representative image shows interplay between two Shigella-septin cages and fused mitochondria. The scale bar represents 1 μm. See also Movie EV3.B HeLa cells were treated with Drp1 siRNA for 72 h, and then infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.C HeLa cells were infected with S. flexneri M90T for 4 h 40 min, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show the length of mitochondrial (μm; whiskers from min to max) surrounding Shigella outside (-) or inside (+) SEPT7 cages from 3 independent experiments (analysis of at least 130 measurements per biological replicate). Student's t-test; *** = p<0.001.D HeLa cells were either kept uninfected as a control or infected with S. flexneri M90T or S. flexneri ΔIcsA for 4 h 40 min. Samples were labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show length of mitochondria (μm; whiskers from min to max) in uninfected cells (CTRL), surrounding S. flexneri M90T (+M90T) or S. flexneri ΔIcsA (+ΔIcsA) from 3 independent experiments (analysis of at least 250 measurements per biological replicate). Student's t-test; *** = p<0.001.


Figure 1CSF PGRN is increased in mutation carriers ( MC ) compared to non - carriers ( NC ) . CSF PGRN is not associated with age in either NC or MC . CSF PGRN is increased in males compare to females . CSF PGRN levels do not differ among MC participants carrying a PSEN1 , PSEN2 or APP mutation .

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Figure 1CSF PGRN is increased in mutation carriers (MC) compared to non-carriers (NC).CSF PGRN is not associated with age in either NC or MC.CSF PGRN is increased in males compare to females.CSF PGRN levels do not differ among MC participants carrying a PSEN1, PSEN2 or APP mutation.


Figure 2CSF PGRN as a function of EYO in mutation carriers ( MC , red ) and non - carriers ( NC , blue ) . The solid lines indicate the regression line for each of the groups and the 95 % confidence interval ( CI ) calculated by a linear model adjusting by gender . The interaction term of mutation status and EYO is significant ( P = 0 . 041 ) , also when including PGRN outliers and participants with EYO > + 20 ( P = 0 . 030 ) . Individual data points are not displayed to prevent disclosure of mutation status . The graph depicts the standardised differences of CSF PGRN between MCs and NCs as a function of EYO , in the context of other biomarker and cognitive changes . The curves were generated by the linear model that best fit each marker ( see statistics section and Appendix Table S2 ) . CSF PGRN is significantly increased in MC compared to NC 10 years before the expected symptom onset ( shadowed area ) after brain amyloidosis and brain injury ( as measured by CSF T - tau ) have started , and shortly before CSF sTREM2 starts to increase . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; CSF , cerebrospinal fluid ; MC , mutation carriers ; MMSE , Mini - Mental State Examination ; NC , non - carriers ; T - tau , total tau .

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Figure 2CSF PGRN as a function of EYO in mutation carriers (MC, red) and non-carriers (NC, blue). The solid lines indicate the regression line for each of the groups and the 95% confidence interval (CI) calculated by a linear model adjusting by gender. The interaction term of mutation status and EYO is significant (P = 0.041), also when including PGRN outliers and participants with EYO > +20 (P = 0.030). Individual data points are not displayed to prevent disclosure of mutation status.The graph depicts the standardised differences of CSF PGRN between MCs and NCs as a function of EYO, in the context of other biomarker and cognitive changes. The curves were generated by the linear model that best fit each marker (see statistics section and Appendix Table S2). CSF PGRN is significantly increased in MC compared to NC 10 years before the expected symptom onset (shadowed area) after brain amyloidosis and brain injury (as measured by CSF T-tau) have started, and shortly before CSF sTREM2 starts to increase. Abbreviations: Aβ1-42: amyloid-β 42; CSF, cerebrospinal fluid; MC, mutation carriers; MMSE, Mini-Mental State Examination; NC, non-carriers; T-tau, total tau.


Figure 3Scatter plot representing the levels of CSF PGRN in healthy controls ( depicted in blue ) and the different stages of the Alzheimer ' s continuum ( depicted in red ) . The analysis and graphs were performed excluding CSF PGRN outliers ( 1 ' healthy control ' , 1 ' Preclinical AD A + / TN - ' , 4 ' AD CDR = 0 . 5 ' and 1 ' AD CDR = 1 ' ) . Including them yielded a similar result and CSF PGRN was still significantly higher in the ' AD CDR = 1 ' group compared to the ' healthy controls ' ( P = 0 . 001 ) and ' Preclinical AD A + / TN - ' ( P = 0 . 0001 ) groups . Abbreviations : A : amyloid - β biomarker status ; AD : Alzheimer ' s disease ; CDR : clinical dementia rating ; CSF , cerebrospinal fluid ; N : neurodegeneration biomarker status ; T : tau pathology biomarker status .

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Figure 3Scatter plot representing the levels of CSF PGRN in healthy controls (depicted in blue) and the different stages of the Alzheimer's continuum (depicted in red). The analysis and graphs were performed excluding CSF PGRN outliers (1 'healthy control', 1 'Preclinical AD A+/TN-', 4 'AD CDR = 0.5' and 1 'AD CDR = 1'). Including them yielded a similar result and CSF PGRN was still significantly higher in the 'AD CDR = 1' group compared to the 'healthy controls' (P = 0.001) and 'Preclinical AD A+/TN-' (P = 0.0001) groups. Abbreviations: A: amyloid-β biomarker status; AD: Alzheimer's disease; CDR: clinical dementia rating; CSF, cerebrospinal fluid; N: neurodegeneration biomarker status; T: tau pathology biomarker status.


Figure 4Scatter plots representing the association of CSF PGRN with different cognitive tests . Only the subjects of the Alzheimer ' s continuum group ( n = 474 ) were included . In all tests studied , higher levels of CSF PGRN were associated with worse cognitive performance ( namely , lower scores in ADNI - Mem , ADNI - EF and MMSE and higher scores in ADAS - Cog11 , ADAS - Cog13 and CDR - SB ) . The analysis and the graphs are excluding PGRN outliers ; including them rendered similar results ( Appendix Table S6 ) . Abbreviations : ADAS - Cog , Alzheimer ' s disease Assessment Scale - cognitive subscale ; ADNI - Mem : ADNI memory composite score ; ADNI - EF : ADNI executive function composite score ; CDR - SB : clinical dementia rating Sum of Boxes ; MMSE , Mini - Mental State Examination .

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Figure 4Scatter plots representing the association of CSF PGRN with different cognitive tests. Only the subjects of the Alzheimer's continuum group (n = 474) were included. In all tests studied, higher levels of CSF PGRN were associated with worse cognitive performance (namely, lower scores in ADNI-Mem, ADNI-EF and MMSE and higher scores in ADAS-Cog11, ADAS-Cog13 and CDR-SB). The analysis and the graphs are excluding PGRN outliers; including them rendered similar results (Appendix Table S6). Abbreviations: ADAS-Cog, Alzheimer's disease Assessment Scale - cognitive subscale; ADNI-Mem: ADNI memory composite score; ADNI-EF: ADNI executive function composite score; CDR-SB: clinical dementia rating Sum of Boxes; MMSE, Mini-Mental State Examination.


Figure 5Scatter plots representing the association of CSF PGRN with temporo - parietal FDG - PET uptake within the subjects of the Alzheimer ' s continuum group ( n = 474 ) . Abbreviations : FDG - PET : Fludeoxyglucose positron emission tomographyScatter plots representing the association of CSF PGRN with total hippocampal volume ( B ) within the subjects of the Alzheimer ' s continuum group ( n = 474 ) .

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Figure 5Scatter plots representing the association of CSF PGRN with temporo-parietal FDG-PET uptake within the subjects of the Alzheimer's continuum group (n = 474). Abbreviations: FDG-PET: Fludeoxyglucose positron emission tomographyScatter plots representing the association of CSF PGRN with total hippocampal volume (B) within the subjects of the Alzheimer's continuum group (n = 474).


Figure 6Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers ( T - tau , P - tau181P and Aβ1 − 42 ) in non - carriers ( NC , blue ; A , C , E and G ) and in mutation carriers ( MC , red ; B , D , F and H ) . Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95 % confidence interval ( CI ) for each of the groups . The standardized regression coefficients ( β ) and the P - values are shown and were computed using a linear model adjusting for age , gender and APOE ε4 . The sample contained some outliers ( defined as 3 SDs below or above the group mean ) of the CSF core markers of AD . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; T - tau : total tau ; P - tau : tau phosphorylated at Threonine 181 .

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 O O O O O O O O O O O O O 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 6Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in non-carriers (NC, blue; A, C, E and G) and in mutation carriers (MC, red; B, D, F and H). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181.


Figure 7Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers ( T - tau , P - tau181P and Aβ1 − 42 ) in healthy controls ( blue ; A , D , G and J ) , Alzheimer ' s continuum ( red ; B , E , H and K ) and ' suspected non - Alzheimer ' s pathophysiology ( SNAP ) ' groups ( green ; C , F , I and L ) . Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95 % confidence interval ( CI ) for each of the groups . The standardized regression coefficients ( β ) and the P - values are shown and were computed using a linear model adjusting for age , gender and APOE ε4 . The sample contained some outliers ( defined as 3 SDs below or above the group mean ) of the CSF core markers of AD , the analysis including these outliers yielded similar results ( Appendix Table S8 ) . The Aβ1 - 42 values used for the associations test are those based on an extrapolation curve since the upper technical limit is 1700pg / ml . We also tested the associations with Aβ1 - 42 values truncated at the upper technical limit and the result was similar . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; T - tau : total tau ; P - tau : tau phosphorylated at Threonine 181 ; SNAP : suspected non - Alzheimer ' s pathophysiology .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in healthy controls (blue; A, D, G and J), Alzheimer's continuum (red; B, E, H and K) and 'suspected non-Alzheimer's pathophysiology (SNAP)' groups (green; C, F, I and L). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD, the analysis including these outliers yielded similar results (Appendix Table S8). The Aβ1-42 values used for the associations test are those based on an extrapolation curve since the upper technical limit is 1700pg/ml. We also tested the associations with Aβ1-42 values truncated at the upper technical limit and the result was similar. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181; SNAP: suspected non-Alzheimer's pathophysiology.


Figure 1 ( A ) Representative Western Blot analysis of the endogenous p150Glued co - immunoprecipitated with EB1 in shCTL or shSTIM1 ECs ( left ) and its quantification by normalized densitometry ( right ) . Negative control ( CTL ) was performed incubating cell lysate with protein A - or G - Sepharose and empty mouse IgG . Results are the average ± SD of three independent assays . shCTL value of each biological replicate was normalized on itself and so shSTIM1 experimental value . Results were analyzed by a parametric two - tailed analysis of variance ( ANOVA ) with Bonferroni post hoc analysis . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL and shSTIM1 p ≤ 0 , 05 * . ( B ) Representative of three Western Blot analysis of endogenous p150Glued , light - intermediate chain ( LIC ) of cytoplasmic dynein 1 and EB1 immunoprecipitated with STIM1 in wild - type ECs . Negative control ( CTL ) was performed incubating cell lysate with protein A - or G - Sepharose and empty rabbit IgG . ( D ) Representative Western Blot analysis of increasing amount of GST - STIM1 ( cytoplasmic domain ) pulled down by Cap - Gly - CC1 - p150Glued - V5 - coated beads , in absence or presence of FLAG - EB1 C - terminal . Colors label the protein as in ( c ) . Negative control was performed using equal amount of empty GST protein , together with Cap - Gly - CC1 - p150Glued . ( E ) Representative Western Blot analysis of mCherry - p150Glued WT co - immunoprecipitated with GFP - STIM1 WT , NN , ΔCC1 - 3 / WT ( ΔCC / WT ) or ΔCC1 - 3 / NN ( ΔCC / NN ) in co - transfected HEK 293T cells . Negative control ( CTL ) was performed incubating cell lysate from HEK 293T co - transfected with an empty GFP vector together with mCherry - P150Glued WT with pre - cleared protein A or G - Sepharose and the rabbit GFP antibody . Below , its quantification by normalized densitometry . Results are the average ± SD of three independent assays . The value of P150Glued co - immunoprecipitated with GFP - STIM1 WT from each biological replicate was normalized on itself and so those immunoprecipitated with GFP - STIM1 NN , ΔCC / WT or ΔCC / NN . Results were analyzed by a parametric two - tailed analysis of variance ( ANOVA ) with Bonferroni post hoc analysis . ANOVA p ≤ 0 , 001 ; Bonferroni for STIM1 WT and NN p > 0 . 05 not significant ( ns ) , for STIM1 WT and ΔCC / WT p > 0 . 05 not significant ( ns ) and for STIM1 WT and ΔCC / NN p ≤ 0 , 05 * . ( F ) Representative Western Blot analysis of GST - STIM1 ( cytoplasmic domain ) pulled down by Cap - Gly - CC1 - p150Glued - V5 - coated beads , in presence of FLAG - EB1 C - terminal WT or ΔY . Negative control was performed using equal amount of empty GST protein , together with Cap - Gly - CC1 - p150Glued . ( G ) Confocal microscopy analysis of wild - type ECs , transiently transfected with GFP - STIM1 WT or , NN or ΔCC1 - 3 / WT ( ΔCC / WT ) together with mCherry - P150Glued . Scale bar = 10 μm . Right insets are shown to highlight MT - bound p150Glued + punctae ( arrows ) , colocalized with STIM1 . Scale bar = 5 μm . ( H ) Average number of the mCherry - P150Glued + punctae in the same cells as in ( g ) . Counts are the average ± SEM of three independent experiments for a total of 150 punctae ( 30 cells ) . Results were analyzed by a parametric two - tailed analysis of variance ( ANOVA ) with Bonferroni post hoc analysis . ANOVA p ≤ 0 , 001 ; Bonferroni for STIM1 WT and NN p ≤ 0 , 001 * * * and for STIM1 WT and ΔCC / WT p > 0 . 05 not significant ( ns ) . ( I ) Co - localization analysis of mCherry - P150Glued with GFP - STIM1 WT , NN or ΔCC1 - 3 / WT ( ΔCC / WT ) , transiently co - transfected in ECs , as in ( g ) . Results are the average ± SEM of three independent experiments for a total of 150 punctae ( 30 cells ) and analyzed by a parametric two - tailed analysis of variance ( ANOVA ) with Bonferroni post hoc analysis . ANOVA p ≤ 0 , 001 ; Bonferroni for STIM1 WT and NN p ≤ 0 , 001 * * * and for STIM1 WT and ΔCC / WT p > 0 . 05 not significant ( ns ) .

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 O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative Western Blot analysis of the endogenous p150Glued co-immunoprecipitated with EB1 in shCTL or shSTIM1 ECs (left) and its quantification by normalized densitometry (right). Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty mouse IgG. Results are the average ± SD of three independent assays. shCTL value of each biological replicate was normalized on itself and so shSTIM1 experimental value. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL and shSTIM1 p≤ 0,05 *. (B) Representative of three Western Blot analysis of endogenous p150Glued, light-intermediate chain (LIC) of cytoplasmic dynein 1 and EB1 immunoprecipitated with STIM1 in wild-type ECs. Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty rabbit IgG. (D) Representative Western Blot analysis of increasing amount of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in absence or presence of FLAG-EB1 C-terminal. Colors label the protein as in (c). Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued.(E) Representative Western Blot analysis of mCherry-p150Glued WT co-immunoprecipitated with GFP-STIM1 WT, NN, ΔCC1-3/WT (ΔCC/WT) or ΔCC1-3/NN (ΔCC/NN) in co-transfected HEK 293T cells. Negative control (CTL) was performed incubating cell lysate from HEK 293T co-transfected with an empty GFP vector together with mCherry-P150Glued WT with pre-cleared protein A or G-Sepharose and the rabbit GFP antibody. Below, its quantification by normalized densitometry. Results are the average ± SD of three independent assays. The value of P150Glued co-immunoprecipitated with GFP-STIM1 WT from each biological replicate was normalized on itself and so those immunoprecipitated with GFP-STIM1 NN, ΔCC/WT or ΔCC/NN. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for STIM1 WT and NN p> 0.05 not significant (ns), for STIM1 WT and ΔCC/WT p> 0.05 not significant (ns) and for STIM1 WT and ΔCC/NN p≤ 0,05 *. (F) Representative Western Blot analysis of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in presence of FLAG-EB1 C-terminal WT or ΔY. Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued.  (G) Confocal microscopy analysis of wild-type ECs, transiently transfected with GFP-STIM1 WT or, NN or ΔCC1-3/WT (ΔCC/WT) together with mCherry-P150Glued. Scale bar = 10 μm. Right insets are shown to highlight MT-bound p150Glued+ punctae (arrows), colocalized with STIM1. Scale bar = 5 μm. (H) Average number of the mCherry-P150Glued+ punctae in the same cells as in (g). Counts are the average ± SEM of three independent experiments for a total of 150 punctae (30 cells). Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for STIM1 WT and NN p≤ 0,001 *** and for STIM1 WT and ΔCC/WT p> 0.05 not significant (ns). (I) Co-localization analysis of mCherry-P150Glued with GFP-STIM1 WT, NN or ΔCC1-3/WT (ΔCC/WT), transiently co-transfected in ECs, as in (g). Results are the average ± SEM of three independent experiments for a total of 150 punctae (30 cells) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for STIM1 WT and NN p≤ 0,001 *** and for STIM1 WT and ΔCC/WT p> 0.05 not significant (ns).