Figure 5A ) Confocal image of wing disc expressing the human tetraspanin CD63 - GFP expressed in the Hh receiving cells colocalizing with Ptc ( Bac . Ptc - cherry ) in vesicle - like structures ( yellow arrows ) along basal cytonemes . B ) In vivo confocal frames of abdominal histoblasts showing colocalization of CD63 - Cherry and Ptc ( Yac . Ptc - GFP ) at punctae moving in anterograde direction along cytonemes ( white arrowheads show trajectory in time - lapse frames ) . C ) Lateral confocal plane of wing disc showing endogenous Ptc colocalizing with the Drosophila tetraspanin Tsp96F - RFP ( orthologous to the mammalian tetraspanin CD9 / CD81 ) ( yellow arrows indicate co - localization in punctate structures ) . D ) Basolateral in vivo imaging of abdominal histoblasts expressing Tsp96F - RFP in the Hh receiving cells shows colocalization with Ptc - GFP at mobile punctae ( yellow arrows ) . E ) Tsp96F - RFP expressed in wing disc Hh - receiving cells also shows localization in basal cytonemes stabilized by Ihog - CFP expression ( yellow arrows indicate Tsp96F - RFP - positive punctate structures ) . F ) Frames showing in vivo localization of Tsp96 - RFP punctae to cytonemes stabilized with Ihog - CFP expressed in the abdominal histoblasts Hh - receiving cells , moving in an anterograde fashion towards the P compartment ( white arrowheads show trajectory in time - lapse frames ) . G ) Confocal imaging of receiving cells expressing the HA tagged Tsp96F construct also shows co - localization with endogenous Ptc at cytonemes ( yellow arrows indicate Tsp96F - HA and Ptc co - localizing punctate structures ) . H ) Western blot against HA - tag of Immuno - Precipitated sample ( IP ) , Depleted Sample ( Dep ) , High Speed Sample ( HSS ) Whole Sample and Control sample from a co - immunoprecipitation experiment isolating GFP of Tsp96F - HA and Ptc - GFP expressing larvae or GFP and Tsp96F - HA expressing larvae as negative control . Arrowhead indicates the positive band corresponding to Tsp96F - HA expected molecular weight ( 30 - 40 kDa ) , present in all samples including the immuno - precipitated one and not present in the negative control sample .

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 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 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 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 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 O O O O O O O O O O O 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 5A) Confocal image of wing disc expressing the human tetraspanin CD63-GFP expressed in the Hh receiving cells colocalizing with Ptc (Bac.Ptc-cherry) in vesicle-like structures (yellow arrows) along basal cytonemes.B) In vivo confocal frames of abdominal histoblasts showing colocalization of CD63-Cherry and Ptc (Yac.Ptc-GFP) at punctae moving in anterograde direction along cytonemes (white arrowheads show trajectory in time-lapse frames).C) Lateral confocal plane of wing disc showing endogenous Ptc colocalizing with the Drosophila tetraspanin Tsp96F-RFP (orthologous to the mammalian tetraspanin CD9/CD81) (yellow arrows indicate co-localization in punctate structures).D) Basolateral in vivo imaging of abdominal histoblasts expressing Tsp96F-RFP in the Hh receiving cells shows colocalization with Ptc-GFP at mobile punctae (yellow arrows).E) Tsp96F-RFP expressed in wing disc Hh-receiving cells also shows localization in basal cytonemes stabilized by Ihog-CFP expression (yellow arrows indicate Tsp96F-RFP-positive punctate structures).F) Frames showing in vivo localization of Tsp96-RFP punctae to cytonemes stabilized with Ihog-CFP expressed in the abdominal histoblasts Hh-receiving cells, moving in an anterograde fashion towards the P compartment (white arrowheads show trajectory in time-lapse frames).G) Confocal imaging of receiving cells expressing the HA tagged Tsp96F construct also shows co-localization with endogenous Ptc at cytonemes (yellow arrows indicate Tsp96F-HA and Ptc co-localizing punctate structures).H) Western blot against HA-tag of Immuno-Precipitated sample (IP), Depleted Sample (Dep), High Speed Sample (HSS) Whole Sample and Control sample from a co-immunoprecipitation experiment isolating GFP of Tsp96F-HA and Ptc-GFP expressing larvae or GFP and Tsp96F-HA expressing larvae as negative control. Arrowhead indicates the positive band corresponding to Tsp96F-HA expected molecular weight (30-40 kDa), present in all samples including the immuno-precipitated one and not present in the negative control sample.


Figure 6A ) 3D reconstruction of wing disc immuno - labelled for endogenous Ptc and dorsally over - expressing the Tsp96F - HA construct , keeping the ventral side of the disc in wild type condition as internal control . the dorsal accumulation of Ptc in very large punctae across the apico - basal axis , but mainly at the basal side of the epithelium . On the right , graphs represent fluorescence values from apical to basal , comparing dorsal and ventral sides and showing a shift of the highest values towards the basal side after Tsp96 - HA expression . B ) Confocal image of a wing disc with a clone expressing Tsp96F - HA and immuno - labelled with anti - Ptc and anti - HA antibodies , which co - localize in the large basolateral punctae ( yellow arrows ) . C ) Electron microscopy image from immunolabelled ( anti - GFP ) Hh - receiving cells of a wing disc expressing Ptc - GFP and Tsp96F - HA . The detailed image shows large sac structures that accumulate several dense MVBs positive labelled for Ptc .

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 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

Figure 6A) 3D reconstruction of wing disc immuno-labelled for endogenous Ptc and dorsally over-expressing the Tsp96F-HA construct, keeping the ventral side of the disc in wild type condition as internal control. the dorsal accumulation of Ptc in very large punctae across the apico-basal axis, but mainly at the basal side of the epithelium. On the right, graphs represent fluorescence values from apical to basal, comparing dorsal and ventral sides and showing a shift of the highest values towards the basal side after Tsp96-HA expression.B) Confocal image of a wing disc with a clone expressing Tsp96F-HA and immuno-labelled with anti-Ptc and anti-HA antibodies, which co-localize in the large basolateral punctae (yellow arrows).C) Electron microscopy image from immunolabelled (anti-GFP) Hh-receiving cells of a wing disc expressing Ptc-GFP and Tsp96F-HA. The detailed image shows large sac structures that accumulate several dense MVBs positive labelled for Ptc.


Figure 7A ) Ptc is mainly secreted in small EVs ( P100K ) from Cl8 cells . B ) Large EVs ( lEVs ) , small EVs ( sEVs ) and very small EVs ( vsEVs ) were analysed in triplicate by Western blotting . sEVs are enriched in Ptc and in the EV - associated proteins Syntaxin1 ( Synt1A ) , Late bloomer ( Lbm ) and Hsp70 . C ) Fractionation of sEVs by Sucrose Density Gradient : Ptc partially co - fractioned with Synt1A , Lbm and Hsp70 . Note that while unprocessed form of co - receptor Ihog does not co - fraction with Ptc , the processed form does in the same subpopulation . D ) Fractionation of sEVs by Size Exclusion Chromatography : Ptc perfectly co - fractioned with Synt1A , Lbm and Hsp70 . In agreement with the density gradient analysis , unprocessed form of co - receptor Ihog does not co - fraction with Ptc in the same subpopulation of sEVs . E ) Effect on EV - associated Ptc secretion after gene silencing on Cl8 cells by dsRNAs against the SNARE proteins Syb and α - Snap , the tetraspanin Tsp96F and the ESCRT components Hrs , Tsg101 , Shrub and Vps4 . Hsp70 in cells and EVs is detected by different exposition time .

O B-PANEL_START 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 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

Figure 7A) Ptc is mainly secreted in small EVs (P100K) from Cl8 cells.B) Large EVs (lEVs), small EVs (sEVs) and very small EVs (vsEVs) were analysed in triplicate by Western blotting. sEVs are enriched in Ptc and in the EV-associated proteins Syntaxin1 (Synt1A), Late bloomer (Lbm) and Hsp70.C) Fractionation of sEVs by Sucrose Density Gradient: Ptc partially co-fractioned with Synt1A, Lbm and Hsp70. Note that while unprocessed form of co-receptor Ihog does not co-fraction with Ptc, the processed form does in the same subpopulation.D) Fractionation of sEVs by Size Exclusion Chromatography: Ptc perfectly co-fractioned with Synt1A, Lbm and Hsp70. In agreement with the density gradient analysis, unprocessed form of co-receptor Ihog does not co-fraction with Ptc in the same subpopulation of sEVs.E) Effect on EV-associated Ptc secretion after gene silencing on Cl8 cells by dsRNAs against the SNARE proteins Syb and α-Snap, the tetraspanin Tsp96F and the ESCRT components Hrs, Tsg101, Shrub and Vps4. Hsp70 in cells and EVs is detected by different exposition time.


Figure 8A ) Projection images of three confocal sections ( 1 μm ) at the basal side of wing discs expressing the Bac constructs for Hh - GFP and Ptc - cherry , used to analyse co - localization between the ligand and the receptor in wild type conditions and after inhibition of vesicle fusion ( Syb RNAi ) , vesicle trafficking ( Hrs RNAi ) or Ptc protein degradation ( Dor RNAi ) . Boxplot shows co - localization rates using the Manders coefficient for proportion of red covered by green in projected images for wing discs temporally expressing RNAi against ESCRT proteins Hrs and Vps4 , SNAREs Syb and α - Snap , and Dor as control for effects after Ptc degradation - blocking , as well as temporally over - expressing the Tsp96F - HA . Central horizontal lines show median values of N = 8 - 12 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding . Note the significant reduction in co - localization rates ( 1 = total coverage of red by green ) after RNAi expression for both ESCRT and SNARE proteins as well as after Tsp96F - HA over - expression , while RNAi expression for Dor presents greater co - localization . B ) Projection images of three confocal sections ( 1 μm ) from the apical side of the wing discs . Quantification of co - localization rates using apical projection images ; central horizontal lines show median values of N = 8 - 12 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers . Note that at the apical side no clear significant reduction in co - localization rates ( 1 = total coverage of red by green ) is observed after RNAi expression or in Tsp96F - HA over - expression . C ) Correlation coefficients between the maximum value of quantified integrated density ( integrating both fluorescence intensity and signal area ) for Ptc - GFP values ( Yac construct ) and distance from the basal side ( - 1 = basal , + 1 = apical ) . Box plot shows quantification data of discs after RNAi expression for the vacuole sorting protein Dor ( necessary for Ptc degradation ) , the ESCRT Hrs , the SNARE Syb as well as after over - expression of the Tsp96F - HA construct and wild type condition . Central horizontal lines show median values of N = 8 - 12 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers . Note the apical localization of Ptc - GFP highest values after degradation impairment ( Dor RNAi ) in contrast to the basal shift resulting from exocytosis deficiency ( Syb RNAi ) .

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 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 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

Figure 8A) Projection images of three confocal sections (1 μm) at the basal side of wing discs expressing the Bac constructs for Hh-GFP and Ptc-cherry, used to analyse co-localization between the ligand and the receptor in wild type conditions and after inhibition of vesicle fusion (Syb RNAi), vesicle trafficking (Hrs RNAi) or Ptc protein degradation (Dor RNAi). Boxplot shows co-localization rates using the Manders coefficient for proportion of red covered by green in projected images for wing discs temporally expressing RNAi against ESCRT proteins Hrs and Vps4, SNAREs Syb and α-Snap, and Dor as control for effects after Ptc degradation-blocking, as well as temporally over-expressing the Tsp96F-HA. Central horizontal lines show median values of N=8-12 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding. Note the significant reduction in co-localization rates (1= total coverage of red by green) after RNAi expression for both ESCRT and SNARE proteins as well as after Tsp96F-HA over-expression, while RNAi expression for Dor presents greater co-localization.B) Projection images of three confocal sections (1 μm) from the apical side of the wing discs. Quantification of co-localization rates using apical projection images; central horizontal lines show median values of N=8-12 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note that at the apical side no clear significant reduction in co-localization rates (1= total coverage of red by green) is observed after RNAi expression or in Tsp96F-HA over-expression.C) Correlation coefficients between the maximum value of quantified integrated density (integrating both fluorescence intensity and signal area) for Ptc-GFP values (Yac construct) and distance from the basal side (-1=basal, +1=apical). Box plot shows quantification data of discs after RNAi expression for the vacuole sorting protein Dor (necessary for Ptc degradation), the ESCRT Hrs, the SNARE Syb as well as after over-expression of the Tsp96F-HA construct and wild type condition. Central horizontal lines show median values of N=8-12, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note the apical localization of Ptc-GFP highest values after degradation impairment (Dor RNAi) in contrast to the basal shift resulting from exocytosis deficiency (Syb RNAi).


Figure 9A ) Confocal images at the lateral plane showing the immunostaining of the medium threshold Hh target Collier ( Col ) of wild type wing discs and discs after inhibition of vesicle fusion ( α - Snap RNAi ) , vesicle trafficking ( Hrs RNAi ) or in over - expression of the Tsp96F - HA . Quantification of gradient lengths ( μm ) using Collier signal and normalized to the wing pouch size , showing the proportion of wing pouch covered by signal ( 1 = total coverage ) . Quantification was performed for wild type condition as well as in temporally regulated RNAi expression for the ESCRT complex proteins Hrs , Tsg101 , Shrub and Vps4 and for the SNARE proteins Syb , Snap24 , α - Snap , as well as in transient over - expression of the Tsp96F - HA . Central horizontal lines show median values of N = 7 - 22 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers . Note a slight but significant increase in the length of Col signal for all treatment compared to the wild type condition . B ) Lateral confocal images and quantification of the extension of immuno - labeled Engrailed as the high threshold Hh target . Quantification shows number of cell diameters covered by signal from the posterior / anterior border marked by Hh - GFP ( Bac construct ) ; central horizontal lines show median values of N = 6 - 10 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers . Note a significant reduction after RNAi expression , suggesting Hh reception impairment and flattening of the signal gradient . C ) Lateral confocal images and quantification of Ptc transcription as a Hh target through immunolabeling of the reporter β − galactosidase ( Ptc - LacZ ) . Middle graph shows the extension covered by signal normalized to wing pouch size , in wild type condition ( N = 6 ) or after ectopic expression of the Tsp96F - HA construct ( N = 9 ) . Green shaded areas show data variability and dotted line gives the average values . Note the greater distance covered after Tsp96F - HA transient expression . Boxplot ( right ) shows distance quantifications with central horizontal lines showing median values of N = 6 - 9 , box showing lower and upper quartiles and whiskers showing the maximum and minimum excluding outliers . Note a significant extension under Tsp96F - HA expression , suggesting again flattening of the signal gradient .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9A) Confocal images at the lateral plane showing the immunostaining of the medium threshold Hh target Collier (Col) of wild type wing discs and discs after inhibition of vesicle fusion (α-Snap RNAi), vesicle trafficking (Hrs RNAi) or in over-expression of the Tsp96F-HA. Quantification of gradient lengths (μm) using Collier signal and normalized to the wing pouch size, showing the proportion of wing pouch covered by signal (1=total coverage). Quantification was performed for wild type condition as well as in temporally regulated RNAi expression for the ESCRT complex proteins Hrs, Tsg101, Shrub and Vps4 and for the SNARE proteins Syb, Snap24, α-Snap, as well as in transient over-expression of the Tsp96F-HA. Central horizontal lines show median values of N=7-22 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a slight but significant increase in the length of Col signal for all treatment compared to the wild type condition.B) Lateral confocal images and quantification of the extension of immuno-labeled Engrailed as the high threshold Hh target. Quantification shows number of cell diameters covered by signal from the posterior/anterior border marked by Hh-GFP (Bac construct); central horizontal lines show median values of N=6-10 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a significant reduction after RNAi expression, suggesting Hh reception impairment and flattening of the signal gradient.C) Lateral confocal images and quantification of Ptc transcription as a Hh target through immunolabeling of the reporter β−galactosidase (Ptc-LacZ). Middle graph shows the extension covered by signal normalized to wing pouch size, in wild type condition (N=6) or after ectopic expression of the Tsp96F-HA construct (N=9). Green shaded areas show data variability and dotted line gives the average values. Note the greater distance covered after Tsp96F-HA transient expression. Boxplot (right) shows distance quantifications with central horizontal lines showing median values of N=6-9 , box showing lower and upper quartiles and whiskers showing the maximum and minimum excluding outliers. Note a significant extension under Tsp96F-HA expression, suggesting again flattening of the signal gradient.


Figure 1BACE1 or APP was immunoprecipitated from mouse brains and blotted with E4 - PHA lectin ( lower ) or anti - BACE1 or APP antibodies ( upper ) . hAPP indicates the APP23 transgenic mouse model for AD . Proteins from mouse brain membrane fractions were treated with or without PNGase F and then immunoblotted for BACE1 or for syntaxin 6 ( loading control ) . LC - MS base peak chromatogram of desialo - alditol N - glycans derived from mouse brain BACE1 . To simplify the results , N - glycans were chemically desialylated before LC - MS analysis . BACE1 - specific glycans , judged by comparison with N - glycan structures from anti - BACE1 IgG ( shown in Supplementary Fig S2C ) , are highlighted by red squares . Asterisks indicate glycans demonstrated by MS / MS analysis to contain a bisecting GlcNAc structure . Numbers in parentheses indicate the charge state .

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 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

Figure 1BACE1 or APP was immunoprecipitated from mouse brains and blotted with E4-PHA lectin (lower) or anti-BACE1 or APP antibodies (upper). hAPP indicates the APP23 transgenic mouse model for AD.Proteins from mouse brain membrane fractions were treated with or without PNGase F and then immunoblotted for BACE1 or for syntaxin 6 (loading control).LC-MS base peak chromatogram of desialo-alditol N-glycans derived from mouse brain BACE1. To simplify the results, N-glycans were chemically desialylated before LC-MS analysis. BACE1-specific glycans, judged by comparison with N-glycan structures from anti-BACE1 IgG (shown in Supplementary Fig S2C), are highlighted by red squares. Asterisks indicate glycans demonstrated by MS/MS analysis to contain a bisecting GlcNAc structure. Numbers in parentheses indicate the charge state.


Figure 2BACE1 from temporal lobe membrane fractions of NC , eAD , or AD patients was immunoprecipitated and blotted with E4 - PHA ( upper ) or anti - BACE1 ( lower ) . The signal intensity of E4 - PHA relative to that of BACE1 was calculated ( n = 10 ) . The graph shows means ± SEM ( * P < 0 . 05 ; one - way ANOVA with post hoc Tukey - Kramer test . P = 0 . 014 for NC versus eAD , P = 0 . 028 for NC versus AD ) .

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Figure 2BACE1 from temporal lobe membrane fractions of NC, eAD, or AD patients was immunoprecipitated and blotted with E4-PHA (upper) or anti-BACE1 (lower). The signal intensity of E4-PHA relative to that of BACE1 was calculated (n = 10). The graph shows means ± SEM (*P < 0.05; one-way ANOVA with post hoc Tukey-Kramer test. P = 0.014 for NC versus eAD, P = 0.028 for NC versus AD).


Figure 3APP metabolites from 3 - month - old mouse brain membrane ( A ) or soluble ( B ) fractions were immunoblotted . The signal intensity was quantified ( n = 4 - 5 ) . All graphs show means ± SEM ( * P < 0 . 05 ; Student ' s t - test for ( A ) βCTF and ( B ) sAPPα , and Mann - Whitney U - test for the others . P = 0 . 386 for APP , P = 0 . 602 for αCTF , P = 0 . 045 for βCTF , P = 0 . 218 for sAPPα , P = 0 . 022 for sAPPβ ) .

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Figure 3APP metabolites from 3-month-old mouse brain membrane (A) or soluble (B) fractions were immunoblotted. The signal intensity was quantified (n = 4-5). All graphs show means ± SEM (*P < 0.05; Student's t-test for (A) βCTF and (B) sAPPα, and Mann-Whitney U-test for the others. P = 0.386 for APP, P = 0.602 for αCTF, P = 0.045 for βCTF, P = 0.218 for sAPPα, P = 0.022 for sAPPβ).


Figure 4Amounts of Aβ40 or Aβ42 in the TBS - soluble or Gu - HCl - extractable fraction from ( A ) 3 - month - old or ( B ) 12 - month - old hAPP / Mgat3 + / + , hAPP / Mgat3 + / − , or hAPP / Mgat3 − / − brains ( n = 5 ) . P = 0 . 015 for TBS Aβ40 , P = 0 . 034 for TBS Aβ42 , P = 0 . 001 and 0 . 012 for Gn - HCl Aβ40 , P = 0 . 024 for Gn - HCl Aβ42 in ( A ) , P = 0 . 040 for Gn - HCl Aβ40 , P = 0 . 047 for Gn - HCl Aβ42 in ( B ) . Amounts of Aβ40 or Aβ42 in the TBS - soluble or Gu - HCl - extractable fraction from 3 - to 4 - month - old Mgat3 + / + or Mgat3 − / − brains ( n = 7 - 8 ) . An outlier value was rejected by the Smirnov - Grubbs ' test ( P < 0 . 05 ) . P = 0 . 045 for Gn - HCl Aβ40 . Immunostaining of Aβ plaques in 12 - month - old mouse brain ( left ) . Scale bar , 300 μm . The number of FSB - stained Aβ plaques in brain sections prepared from 12 - month - old male mice was quantified ( right ) ( n = 6 ) . P = 0 . 004 for hippocampus , P = 0 . 0003 for cortex . The Y - maze test was performed using 12 - month - old male Mgat3 + / + , Mgat3 − / − , hAPP / Mgat3 + / + , or hAPP / Mgat3 − / − mice ( n = 8 - 10 ) . * * P = 0 . 004 for Mgat3 + / + versus hAPP / Mgat3 + / + , * P = 0 . 022 for hAPP / Mgat3 + / + versus hAPP / Mgat3 − / − .

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 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 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 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

Figure 4Amounts of Aβ40 or Aβ42 in the TBS-soluble or Gu-HCl-extractable fraction from (A) 3-month-old or (B) 12-month-old hAPP/Mgat3+/+, hAPP/Mgat3+/−, or hAPP/Mgat3−/− brains (n = 5). P = 0.015 for TBS Aβ40, P = 0.034 for TBS Aβ42, P = 0.001 and 0.012 for Gn-HCl Aβ40, P = 0.024 for Gn-HCl Aβ42 in (A), P = 0.040 for Gn-HCl Aβ40, P = 0.047 for Gn-HCl Aβ42 in (B).Amounts of Aβ40 or Aβ42 in the TBS-soluble or Gu-HCl-extractable fraction from 3- to 4-month-old Mgat3+/+ or Mgat3−/− brains (n = 7-8). An outlier value was rejected by the Smirnov-Grubbs' test (P < 0.05). P = 0.045 for Gn-HCl Aβ40.Immunostaining of Aβ plaques in 12-month-old mouse brain (left). Scale bar, 300 μm. The number of FSB-stained Aβ plaques in brain sections prepared from 12-month-old male mice was quantified (right) (n = 6). P = 0.004 for hippocampus, P = 0.0003 for cortex.The Y-maze test was performed using 12-month-old male Mgat3+/+, Mgat3−/−, hAPP/Mgat3+/+, or hAPP/Mgat3−/− mice (n = 8-10). **P = 0.004 for Mgat3+/+ versus hAPP/Mgat3+/+, *P = 0.022 for hAPP/Mgat3+/+ versus hAPP/Mgat3−/−.


Figure 5BACE1 from 2 - week - old mouse brains was immunoprecipitated and immunoblotted ( left ) . Immunoprecipitated BACE1 activity was measured in vitro ( right , n = 3 ) . The values of Bace1 − / − samples were subtracted as a background . N . S . , P = 0 . 101 . MEF homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1 , APP , rab5 , or rab7 . EE , early endosome ; LE , late endosome . Brain homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1 , rab5 , or rab9 . EE , early endosome ; LE , late endosome . Signal intensities were quantified and are shown in the right graphs . Immunostaining of 12 - month - old mouse cerebral cortex for BACE1 and APP . A typical image in the vicinity of plaque - forming area is shown for hAPP / Mgat3 + / + brain . The area of co - localization was quantified using random images of cerebral cortex ( n = 10 ) . * P = 0 . 015 . Immunoblot of BACE1 or GAPDH ( loading control ) from immortalized MEFs treated with a proteasome inhibitor ( MG132 ) or a lysosome inhibitor ( chloroquine ; CQ ) . Immunostaining of 12 - month - old mouse cerebral cortex for BACE1 ( F ) , or nicastrin ( G ) , and Lamp1 . LE , late endosome . Scale bar , 10 μm . The area in which co - localized staining was observed was quantified as a percentage of the total BACE1 - positive area ( right , n = 10 ) . * * P = 0 . 007 .

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 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 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 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 5BACE1 from 2-week-old mouse brains was immunoprecipitated and immunoblotted (left). Immunoprecipitated BACE1 activity was measured in vitro (right, n = 3). The values of Bace1−/− samples were subtracted as a background. N.S., P = 0.101.MEF homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1, APP, rab5, or rab7. EE, early endosome; LE, late endosome.Brain homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1, rab5, or rab9. EE, early endosome; LE, late endosome. Signal intensities were quantified and are shown in the right graphs.Immunostaining of 12-month-old mouse cerebral cortex for BACE1 and APP. A typical image in the vicinity of plaque-forming area is shown for hAPP/Mgat3+/+ brain. The area of co-localization was quantified using random images of cerebral cortex (n = 10). *P = 0.015.Immunoblot of BACE1 or GAPDH (loading control) from immortalized MEFs treated with a proteasome inhibitor (MG132) or a lysosome inhibitor (chloroquine; CQ).Immunostaining of 12-month-old mouse cerebral cortex for BACE1 (F), or nicastrin (G), and Lamp1. LE, late endosome. Scale bar, 10 μm. The area in which co-localized staining was observed was quantified as a percentage of the total BACE1-positive area (right, n = 10). **P = 0.007.


Figure 6Immunostaining of 12 - month - old brain sections ( cerebral cortex ) with anti - BACE1 . Scale bar , 100 μm . Proteins from the membrane fractions of 12 - month - old mouse brains ( n = 4 - 5 ) were immunoblotted for BACE1 , and BACE1 intensity was quantified . * P = 0 . 044 . Lysates from MEFs were immunoblotted for APP , BACE1 , or GAPDH ( loading control ) , and BACE1 intensity was quantified ( n = 4 ) . Amount of Aβ40 in the culture medium of immortalized MEFs ( n = 3 ) . * P = 0 . 045 . MEFs were transfected with control siRNA or GGA3 - siRNA and then immunoblotted for BACE1 , GGA3 , or GAPDH ( loading control ) ( n = 6 ) .

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 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 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 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

Figure 6Immunostaining of 12-month-old brain sections (cerebral cortex) with anti-BACE1. Scale bar, 100 μm.Proteins from the membrane fractions of 12-month-old mouse brains (n = 4-5) were immunoblotted for BACE1, and BACE1 intensity was quantified. *P = 0.044.Lysates from MEFs were immunoblotted for APP, BACE1, or GAPDH (loading control), and BACE1 intensity was quantified (n = 4).Amount of Aβ40 in the culture medium of immortalized MEFs (n = 3). *P = 0.045.MEFs were transfected with control siRNA or GGA3-siRNA and then immunoblotted for BACE1, GGA3, or GAPDH (loading control) (n = 6).


Figure 7Membrane fractions from 3 - week - old Mgat3 + / + , Mgat3 − / − , and Bace1 − / − mice were immunoblotted for CHL1 ( upper ) , contactin - 2 ( middle ) , or syntaxin 6 ( lower ) ( n = 4 - 5 ) . The graphs show means ± SEM ( * * P < 0 . 01 ; two - way ANOVA with post hoc Tukey - Kramer test , P = 0 . 006 for Mgat3 + / + versus Bace1 − / − , P = 0 . 008 for Mgat3 − / − versus Bace1 − / − in CHL1 / syn6 , P = 0 . 001 for Mgat3 + / + versus Bace1 − / − , P = 0 . 002 for Mgat3 − / − versus Bace1 − / − in Contactin - 2 / syn6 ) . Number of pups surviving at 4 weeks following a cross between heterozygous male and female mice .

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Figure 7Membrane fractions from 3-week-old Mgat3+/+, Mgat3−/−, and Bace1−/− mice were immunoblotted for CHL1 (upper), contactin-2 (middle), or syntaxin 6 (lower) (n = 4-5). The graphs show means ± SEM (**P < 0.01; two-way ANOVA with post hoc Tukey-Kramer test, P = 0.006 for Mgat3+/+ versus Bace1−/−, P = 0.008 for Mgat3−/− versus Bace1−/− in CHL1/syn6, P = 0.001 for Mgat3+/+ versus Bace1−/−, P = 0.002 for Mgat3−/− versus Bace1−/− in Contactin-2/syn6).Number of pups surviving at 4 weeks following a cross between heterozygous male and female mice.


Figure 1 ( A ) Heteroplasmy levels by PCR - RFLP at position 3243 in 82 hiPSCs clones at passage 2 , derived from three MELAS patients ( MitoA , MitoB , MitoC ) . The black dots represent the mutation load for a specific hiPSC clone ; n is the total number of individual hiPSC clones analyzed per patient . The red stars represent the heteroplasmy levels for the parental fibroblasts . ( A ) Heteroplasmy levels by PCR - RFLP at position 3243 in 82 hiPSCs clones at passage 2 , derived from three MELAS patients ( MitoA , MitoB , MitoC ) . The black dots represent the mutation load for a specific hiPSC clone ; n is the total number of individual hiPSC clones analyzed per patient . The red stars represent the heteroplasmy levels for the parental fibroblasts .

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 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

Figure 1(A) Heteroplasmy levels by PCR-RFLP at position 3243 in 82 hiPSCs clones at passage 2, derived from three MELAS patients (MitoA, MitoB, MitoC). The black dots represent the mutation load for a specific hiPSC clone; n is the total number of individual hiPSC clones analyzed per patient. The red stars represent the heteroplasmy levels for the parental fibroblasts.(A) Heteroplasmy levels by PCR-RFLP at position 3243 in 82 hiPSCs clones at passage 2, derived from three MELAS patients (MitoA, MitoB, MitoC). The black dots represent the mutation load for a specific hiPSC clone; n is the total number of individual hiPSC clones analyzed per patient. The red stars represent the heteroplasmy levels for the parental fibroblasts.


Figure 2 ( A ) mtDNA NGS of MitoA fibroblasts ( MitoA FB at passage 5 ) and MitoA - derived hiPSCs ( passages 7 - 9 ) ; n = 13 . ( B ) mtDNA NGS of MitoB fibroblasts ( MitoB FB at passage 3 ) and MitoB - derived hiPSCs ( passages 5 - 11 ) ; n = 12 . ( C ) mtDNA NGS of MitoC fibroblasts ( MitoC FB at passage 7 ) and MitoC - derived hiPSCs ( passages 5 - 11 ) ; n = 11 . GPMs are represented by black diamonds at the specific mtDNA positions ; small black diamonds denote mutation load between 1 and 10 % , medium black diamonds 10 to 40 % , and large black diamonds > 40 % . Red diamonds : point mutation A3243G ( MELAS causing disease mutation ) . Numbers beside diamonds indicate the specific mutation load at that position .

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

Figure 2(A) mtDNA NGS of MitoA fibroblasts (MitoA FB at passage 5) and MitoA-derived hiPSCs (passages 7-9); n=13.(B) mtDNA NGS of MitoB fibroblasts (MitoB FB at passage 3) and MitoB-derived hiPSCs (passages 5-11); n=12.(C) mtDNA NGS of MitoC fibroblasts (MitoC FB at passage 7) and MitoC-derived hiPSCs (passages 5-11); n=11.GPMs are represented by black diamonds at the specific mtDNA positions; small black diamonds denote mutation load between 1 and 10%, medium black diamonds 10 to 40%, and large black diamonds > 40%. Red diamonds: point mutation A3243G (MELAS causing disease mutation). Numbers beside diamonds indicate the specific mutation load at that position.


Figure 3 ( B ) Distribution of GPMs with mutation load lower or greater than 10 % , in control and MELAS groups . The majority of GPMs are present at levels lower than 10 % in either group . ( C ) Location of GPMs greater than 10 % , in mtDNA .

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Figure 3(B) Distribution of GPMs with mutation load lower or greater than 10%, in control and MELAS groups. The majority of GPMs are present at levels lower than 10% in either group.(C) Location of GPMs greater than 10%, in mtDNA.


Figure 4 ( B ) mtDNA NGS showing the position and mutation load of GPMs in seven isogenic MitoA - derived hiPSCs clones and their differentiated counterparts . The mtDNA genotype is stable during in vitro cardiac differentiation and lactate enrichment . MitoA hiPSCs 57 and 65 ( wild - type ) are labelled in black , MitoA hiPSCs 61 and 69 ( m . 3243A > G mutant ) are labelled in red and MitoA hiPSCs 58 , 59 and 217 ( with amplified GPMs in nuclear reprogramming ) are labelled in blue . ( C ) Quantification of the percentage of mature hiPSCs - derived cardiomyocytes in direct differentiation of hiPSC clones . Representative histograms showing the percentage of the cells expressing mature cardiac markers : myosin heavy chain ( MF20 ) , troponin T ( cTnT ) and troponin I ( cTnI ) by flow cytometry , in MitoA - 57 - derived cardiomyocytes ( wild - type ) and MitoA - 69 - derived cardiomyocytes ( 83 % m . 3243A > G ) . For complete quantification of cardiac markers in all hiPSCs - derived cardiomyocytes generated . ( D ) Immunofluorescence images of cardiac markers ( cTnT and MF20 ) showing similar sarcomere structures in hiPSC - derived cardiomyocytes wild - type ( MitoA - 57 ) and mutant ( MitoA - 69 ) . ( E ) Oxygen consumption rate ( OCR ) in hiPSCs . OCR reported as a % of that in MitoA - 57 hiPSC , the control line . MitoA - 57 and MitoA - 65 hiPSCs wild - type ( in black ) , MitoA - 61 and MitoA - 69 m . 3243A > G mutant ( in red ) , MitoA - 58 , MitoA - 59 and MitoA - 217 hiPSCs amplified GPMs ( in blue ) . Error bars are mean ± SD and OCR data are representative 3 biological replicates in all samples . ( F ) Oxygen consumption rate in hiPSCs - derived cardiomyocytes at day 19 of cardiac differentiation . OCR reported as % of that in MitoA - 57 - derived cardiomyocytes , the control line . MitoA - 57 and MitoA - 65 derived cardiomyocytes wild - type ( in black ) , MitoA - 61 and MitoA - 69 derived cardiomyocytes mutants m . 3243A > G ( in red ) , MitoA - 58 , MitoA - 59 and MitoA - 217 derived cardiomyocytes with amplified GPMs ( in blue ) . Error bars are mean ± SD and OCR data are representative of two independent in vitro cardiac differentiation experiments , with at least 3 biological replicates in each sample , and significance established using Student ' s t - 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 O O O O O O O O O O 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 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

Figure 4(B) mtDNA NGS showing the position and mutation load of GPMs in seven isogenic MitoA-derived hiPSCs clones and their differentiated counterparts. The mtDNA genotype is stable during in vitro cardiac differentiation and lactate enrichment. MitoA hiPSCs 57 and 65 (wild-type) are labelled in black, MitoA hiPSCs 61 and 69 (m.3243A>G mutant) are labelled in red and MitoA hiPSCs 58, 59 and 217 (with amplified GPMs in nuclear reprogramming) are labelled in blue.(C) Quantification of the percentage of mature hiPSCs-derived cardiomyocytes in direct differentiation of hiPSC clones. Representative histograms showing the percentage of the cells expressing mature cardiac markers: myosin heavy chain (MF20), troponin T (cTnT) and troponin I (cTnI) by flow cytometry, in MitoA-57-derived cardiomyocytes (wild-type) and MitoA-69-derived cardiomyocytes (83% m.3243A>G). For complete quantification of cardiac markers in all hiPSCs-derived cardiomyocytes generated.(D) Immunofluorescence images of cardiac markers (cTnT and MF20) showing similar sarcomere structures in hiPSC-derived cardiomyocytes wild-type (MitoA-57) and mutant (MitoA-69).(E) Oxygen consumption rate (OCR) in hiPSCs. OCR reported as a % of that in MitoA-57 hiPSC, the control line. MitoA-57 and MitoA-65 hiPSCs wild-type (in black), MitoA-61 and MitoA-69 m.3243A>G mutant (in red), MitoA-58, MitoA-59 and MitoA-217 hiPSCs amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative 3 biological replicates in all samples.(F) Oxygen consumption rate in hiPSCs-derived cardiomyocytes at day 19 of cardiac differentiation. OCR reported as % of that in MitoA-57-derived cardiomyocytes, the control line. MitoA-57 and MitoA-65 derived cardiomyocytes wild-type (in black), MitoA-61 and MitoA-69 derived cardiomyocytes mutants m.3243A>G (in red), MitoA-58, MitoA-59 and MitoA-217 derived cardiomyocytes with amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative of two independent in vitro cardiac differentiation experiments, with at least 3 biological replicates in each sample, and significance established using Student's t-test.


Figure 1B . Representative E - cadherin ( E - cad ) and DAPI immunofluorescence images of AECs treated apically with TGFβ1 or vehicle control after 72 h in the co - culture system ( scale bars 25 µm ) . E . ELISA analysis shows increased αSMA levels in a TGFβ1 dose - dependent manner from MCs and IPF - MCs in the co - culture system . Note a stronger αSMA response in IPF - MCs ( mean + s . d . , n = 3 biological replicates from 6 MCs and 6 IPF - MCs donors , * * p = 0 . 0014 , * * * * p < 0 . 0001 , Nonlinear Regression followed by ANOVA / Sidak ' s ) . G , H . 72 h after substitutions in the co - culture , αSMA and E - cad levels of MCs and AECs , respectively , were measured with ELISA and normalised to the control . Note replacement of injured AECs with untreated AECs or replacement of media and MCs attenuates αSMA expression and increases E - cad levels ( mean + s . d . , ns = non - significant , * * * p < 0 . 001 , * * p < 0 . 01 , n = 3 biological replicates with 6 MCs donors , repeated measurement ANOVA for MCs or ordinary ANOVA for AECs with Dunnett ´ s posthoc test ) .

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Figure 1B. Representative E-cadherin (E-cad) and DAPI immunofluorescence images of AECs treated apically with TGFβ1 or vehicle control after 72 h in the co-culture system (scale bars 25 µm).E. ELISA analysis shows increased αSMA levels in a TGFβ1 dose-dependent manner from MCs and IPF-MCs in the co-culture system. Note a stronger αSMA response in IPF-MCs (mean + s.d., n = 3 biological replicates from 6 MCs and 6 IPF-MCs donors, **p = 0.0014, ****p < 0.0001, Nonlinear Regression followed by ANOVA/Sidak's).G, H. 72 h after substitutions in the co-culture, αSMA and E-cad levels of MCs and AECs, respectively, were measured with ELISA and normalised to the control. Note replacement of injured AECs with untreated AECs or replacement of media and MCs attenuates αSMA expression and increases E-cad levels (mean + s.d., ns = non-significant, ***p < 0.001, **p < 0.01, n = 3 biological replicates with 6 MCs donors, repeated measurement ANOVA for MCs or ordinary ANOVA for AECs with Dunnett´s posthoc test).


Figure 2B . Volcano plot representing logarithmic ratio of differentially secreted proteins from proteomics analysis of co - culture medium upon apical TGFβ1 stimulation ( p - adj < 0 . 05 , t - test ) , with examples of profibrotic secreted proteins ( red dots indicating significantly upregulated proteins , blue dots indicating significantly downregulated proteins , and grey dots indicating no significant change in protein expression levels ) . H . Representative H3K27me3 and EZH2 immunofluorescence images and box plots ( minimum , first quartile , median , third quartile and maximum ) showing decreased H3K27me3 levels but increased total EZH2 levels in TGFβ1 - injured AECs in co - culture with MCs ( n = 5 biological replicates with > 50 cells per experiment , scale bars 50 µm , * p < 0 . 05 , unpaired t - test ) .

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Figure 2B. Volcano plot representing logarithmic ratio of differentially secreted proteins from proteomics analysis of co-culture medium upon apical TGFβ1 stimulation (p-adj < 0.05, t-test), with examples of profibrotic secreted proteins (red dots indicating significantly upregulated proteins, blue dots indicating significantly downregulated proteins, and grey dots indicating no significant change in protein expression levels).H. Representative H3K27me3 and EZH2 immunofluorescence images and box plots (minimum, first quartile, median, third quartile and maximum) showing decreased H3K27me3 levels but increased total EZH2 levels in TGFβ1-injured AECs in co-culture with MCs (n = 5 biological replicates with >50 cells per experiment, scale bars 50 µm, *p < 0.05, unpaired t-test).


Figure 3G . EZH2 - deleted AECs ( sgEZH2 ) reintroducing empty vector ( EV ) , T311 wildtype ( WT ) , a phosphorylated - deficient T311A , or a phosphomimetic T311D form of EZH2 were quantified for the expression of profibrotic genes . Vehicle and TGFβ1 treated AECs ( sgNEG + vehicle / TGFβ1 ) were used as control . mRNA levels are normalised to HPRT1 expression . ( mean + s . d . , n = 4 biological replicates , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , Kruskal - Wallis / Dunn ' s ) .

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Figure 3G. EZH2-deleted AECs (sgEZH2) reintroducing empty vector (EV), T311 wildtype (WT), a phosphorylated-deficient T311A, or a phosphomimetic T311D form of EZH2 were quantified for the expression of profibrotic genes. Vehicle and TGFβ1 treated AECs (sgNEG + vehicle / TGFβ1) were used as control. mRNA levels are normalised to HPRT1 expression. (mean + s.d., n = 4 biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis/Dunn's).


Figure 4B . Nuclear fractionation followed by simple western analysis ( Peggy Sue ) of AECs exposed to 72 h of TGFβ1 in the co - culture system shows an increase in phosphorylated TAK1 and a parallel increase in ph - EZH2 . Quantifications ( lower panels ) show mean + s . d . , n = 3 biological replicates ( ns = non - significant , * * * p < 0 . 001 , Kruskal - Wallis / Dunn ' s ) . E . ChIP - qPCR shows diminished POL2 occupancy on profibrotic genes in injured AECs subjected to 5 - OZ ( mean + s . d . , n = 3 biological replicates ) . Unspecific IgG was used as negative control .

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Figure 4B. Nuclear fractionation followed by simple western analysis (Peggy Sue) of AECs exposed to 72 h of TGFβ1 in the co-culture system shows an increase in phosphorylated TAK1 and a parallel increase in ph-EZH2. Quantifications (lower panels) show mean + s.d., n = 3 biological replicates (ns = non-significant, ***p < 0.001, Kruskal-Wallis/Dunn's).E. ChIP-qPCR shows diminished POL2 occupancy on profibrotic genes in injured AECs subjected to 5-OZ (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control.


Figure 5C , D . ChIP - qPCR shows increased occupancy of ( C ) POL2 - S5p at promoters of profibrotic genes in AECs subjected to TGFβ1 for 24 h , whereas no enrichment of ( D ) POL2 - S2p at the gene bodies of these genes was detected . Negative IgG control is shown in Appendix Figure S5B ( mean + s . d . , n = 3 biological replicates ) . E , F . ChIP - qPCR shows increased occupancy of ( E ) POL2 - S5p at promoters and ( F ) POL2 - S2p at the gene bodies of profibrotic genes in AECs subjected to TGFβ1 for 48 h . Negative IgG control is shown in Appendix Figure S5C ( mean + s . d . , n = 3 biological replicates ) .

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Figure 5C, D. ChIP-qPCR shows increased occupancy of (C) POL2-S5p at promoters of profibrotic genes in AECs subjected to TGFβ1 for 24 h, whereas no enrichment of (D) POL2-S2p at the gene bodies of these genes was detected. Negative IgG control is shown in Appendix Figure S5B (mean + s.d., n = 3 biological replicates). E, F. ChIP-qPCR shows increased occupancy of (E) POL2-S5p at promoters and (F) POL2-S2p at the gene bodies of profibrotic genes in AECs subjected to TGFβ1 for 48 h. Negative IgG control is shown in Appendix Figure S5C (mean + s.d., n = 3 biological replicates).


Figure 6B . Simple western analysis ( Peggy Sue ) of EZH2 immunoprecipitates shows abolition of TGFβ1 - induced profibrotic transcriptional complex of EZH2 / POL2 / actin upon the convergent treatment of TGFβ1 and Y27632 . Unspecific IgG binding was used as a negative control . Representative from 3 biological replicates is shown . E . qPCR analysis of profibrotic genes in MCs co - culture with AECs shows that depletion of IPO9 in TGFβ1 - injured AECs blocks the fibrotic crosstalk with MCs . Data show mRNA levels of profibrotic genes normalised to S26 ( mean + s . d . , n = 3 biological replicates with 5 MCs donors , * p < 0 . 05 , * * * p < 0 . 001 , ANOVA / Tukey ' s ) .

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Figure 6B. Simple western analysis (Peggy Sue) of EZH2 immunoprecipitates shows abolition of TGFβ1-induced profibrotic transcriptional complex of EZH2/POL2/actin upon the convergent treatment of TGFβ1 and Y27632. Unspecific IgG binding was used as a negative control. Representative from 3 biological replicates is shown.E. qPCR analysis of profibrotic genes in MCs co-culture with AECs shows that depletion of IPO9 in TGFβ1-injured AECs blocks the fibrotic crosstalk with MCs. Data show mRNA levels of profibrotic genes normalised to S26 (mean + s.d., n = 3 biological replicates with 5 MCs donors, *p < 0.05, ***p < 0.001, ANOVA/Tukey's).


Figure 7A . Simple western analysis ( Peggy Sue ) and quantifications ( right panels ) of mouse lung epithelial cells shows increased ph - EZH2 ( T311 ) , ph - TAK1 , myosin activity ( ph - MLC2 ) and POL2 - K7m levels in AAV - mediated TGFβ1 over - expression . Note , increased ph - EZH2 and POL2 - K7m levels are attenuated by the EZH2 inhibitor GSK126 , whereas ph - TAK1 and ph - MLC2 levels cannot be rescued by GSK126 . Quantifications ( right panels ) show violin plots , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = non - significant , Kruskal - Wallis / Dunn ' s . Data information : All violin plots display minimum , first quartile , median , third quartile and maximum ; n = 5 control , 12 AAV - TGFβ1 and 13 GSK126 - treated AAV - TGFβ1 mice . B . Representative of 3D computed tomography ( CT ) reconstruction of the lung from control , AAV - TGFβ1 and GSK126 - treated AAV - TGFβ1 mice ( green : lung tissue , red : airways , and region - of - interest ( ROI ) : blue ) . Insets show µCT slices in the middle of the lung from respective mice . Note , GSK126 attenuates TGFβ1 - induced lung injury . Quantification ( right panel ) shows mean intensity of ROIs from the whole lung ( violin plots , * p = 0 . 0385 , * * p = 0 . 0012 , ANOVA / Holm - Sidak ' s ) . Data information : All violin plots display minimum , first quartile , median , third quartile and maximum ; n = 5 control , 12 AAV - TGFβ1 and 13 GSK126 - treated AAV - TGFβ1 mice . D . Immunofluorescence analysis of KRT5 as a marker for alveolar metaplastic basal cells and ph - EZH2 ( scale bars 100 µm ) , pro - SFTPC as a marker for alveolar type 2 epithelial cells ( scale bars 50 µm ) and quantifications ( right panels ) show percentage of KRT5 + pods area per 10X field and percentage of pro - SFTPC + cells per 20X field ( * p < 0 . 05 , unpaired t - test ) . Data information : All violin plots display minimum , first quartile , median , third quartile and maximum ; n = 5 control , 12 AAV - TGFβ1 and 13 GSK126 - treated AAV - TGFβ1 mice .

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Figure 7A. Simple western analysis (Peggy Sue) and quantifications (right panels) of mouse lung epithelial cells shows increased ph-EZH2 (T311), ph-TAK1, myosin activity (ph-MLC2) and POL2-K7m levels in AAV-mediated TGFβ1 over-expression. Note, increased ph-EZH2 and POL2-K7m levels are attenuated by the EZH2 inhibitor GSK126, whereas ph-TAK1 and ph-MLC2 levels cannot be rescued by GSK126. Quantifications (right panels) show violin plots, *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant, Kruskal-Wallis/Dunn's. Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.B. Representative of 3D computed tomography (CT) reconstruction of the lung from control, AAV-TGFβ1 and GSK126-treated AAV-TGFβ1 mice (green: lung tissue, red: airways, and region-of-interest (ROI): blue). Insets show µCT slices in the middle of the lung from respective mice. Note, GSK126 attenuates TGFβ1-induced lung injury. Quantification (right panel) shows mean intensity of ROIs from the whole lung (violin plots, *p = 0.0385, **p = 0.0012, ANOVA/Holm-Sidak's). Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.D. Immunofluorescence analysis of KRT5 as a marker for alveolar metaplastic basal cells and ph-EZH2 (scale bars 100 µm), pro-SFTPC as a marker for alveolar type 2 epithelial cells (scale bars 50 µm) and quantifications (right panels) show percentage of KRT5+ pods area per 10X field and percentage of pro-SFTPC+ cells per 20X field (*p < 0.05, unpaired t-test). Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.


f1 ( a ) MEFs stably expressing EGFP - Parkin were exposed to CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis ( IB ) with antibodies to the indicated proteins . The asterisk indicates a nonspecific band . ( b ) SH - SY5Y cells were exposed to CCCP ( 10 μM ) for the indicated times and then subjected to immunoblot analysis . ( c ) MEFs stably expressing EGFP - Parkin were treated with CCCP ( 30 μM ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c and with a confocal microscope . The fluorescence of EGFP - Parkin was monitored directly . ( d ) Higher - magnification views of the boxed areas in c . Scale bar ( c , d ) , 10 μm .

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f1(a) MEFs stably expressing EGFP-Parkin were exposed to CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis (IB) with antibodies to the indicated proteins. The asterisk indicates a nonspecific band.(b) SH-SY5Y cells were exposed to CCCP (10 μM) for the indicated times and then subjected to immunoblot analysis.(c) MEFs stably expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c and with a confocal microscope. The fluorescence of EGFP-Parkin was monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.


f2 ( a , c ) MEFs ( a ) stably expressing FLAG - Parkin and either EGFP - VAP - A or HA - VAP - A were treated with CCCP ( 30 and 10 μM , respectively ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to HA or to FLAG ( M2 ) . The fluorescence of EGFP - VAP - A was also monitored directly . ( b , d ) Higher - magnification views of the boxed areas in a and c , respectively . ( a , c ) HeLa cells ( c ) stably expressing FLAG - Parkin and either EGFP - VAP - A or HA - VAP - A were treated with CCCP ( 30 and 10 μM , respectively ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to HA or to FLAG ( M2 ) . The fluorescence of EGFP - VAP - A was also monitored directly . ( b , d ) Higher - magnification views of the boxed areas in a and c , respectively . ( e ) NIH 3T3 cells stably expressing EGFP - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , after which whole - cell homogenates as well as cytosolic , ER and mitochondrial fractions were prepared and subjected to immunoblot analysis . Scale bar ( a - d ) , 10 μm .

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f2(a,c) MEFs (a) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively.(a,c) HeLa cells (c) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively.(e) NIH 3T3 cells stably expressing EGFP-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, after which whole-cell homogenates as well as cytosolic, ER and mitochondrial fractions were prepared and subjected to immunoblot analysis. Scale bar (a-d), 10 μm.


f3 ( a ) MEFs stably expressing EGFP - tagged wild - type ( WT ) or T240R mutant forms of Parkin as well as FLAG - tagged VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to FLAG ( M2 ) and to cytochrome c . The fluorescence of EGFP was also monitored directly . ( b ) MEFs as in a were treated with CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis . ( c ) MEFs stably expressing EGFP - Parkin and FLAG - VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) and lactacystin ( 10 μM ) for 24 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to FLAG ( M2 ) and to cytochrome c . The fluorescence of EGFP - Parkin was also monitored directly . ( d ) MEFs stably expressing EGFP - Parkin were incubated for 12 h in the absence or presence of CCCP ( 30 μM ) and lactacystin ( 10 μM ) as indicated and then subjected to immunoblot analysis . Scale bar ( a , c ) , 10 μm .

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f3(a) MEFs stably expressing EGFP-tagged wild-type (WT) or T240R mutant forms of Parkin as well as FLAG-tagged VAP-A were incubated in the absence or presence of CCCP (30 μM) for 24 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to FLAG (M2) and to cytochrome c. The fluorescence of EGFP was also monitored directly.(b) MEFs as in a were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis.(c) MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) and lactacystin (10 μM) for 24 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to FLAG (M2) and to cytochrome c. The fluorescence of EGFP-Parkin was also monitored directly.(d) MEFs stably expressing EGFP-Parkin were incubated for 12 h in the absence or presence of CCCP (30 μM) and lactacystin (10 μM) as indicated and then subjected to immunoblot analysis. Scale bar (a,c), 10 μm.


f4 ( b ) MEFs stably expressing FLAG - Parkin and EGFP - tagged forms of FKBP38 , Omp25 , Omp25 / FKBP38 or FKBP38 / Omp25 were treated with CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP was also monitored directly . Scale bar , 10 μm . ( c ) MEFs stably expressing mCherry - tagged Parkin and EGFP - tagged forms of FKBP38 , Omp25 , Omp25 / FKBP38 or FKBP38 / Omp25 were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis .

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f4(b) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were treated with CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. Scale bar, 10 μm.(c) MEFs stably expressing mCherry-tagged Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.


f5 ( a ) MEFs stably expressing FLAG - Parkin and either EGFP - Bcl - 2 or EGFP - Bcl - xL were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis . ( c ) MEFs stably expressing FLAG - Parkin and EGFP - tagged forms of Bcl - 2 , Bcl - xL , Bcl - 2 / xL or Bcl - xL / 2 were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP was also monitored directly . ( d ) Higher - magnification views of the boxed areas in c . Scale bar ( c , d ) , 10 μm .

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f5(a) MEFs stably expressing FLAG-Parkin and either EGFP-Bcl-2 or EGFP-Bcl-xL were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.(c) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of Bcl-2, Bcl-xL, Bcl-2/xL or Bcl-xL/2 were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.


f7 ( a ) MEFs stably expressing EGFP - FKBP38 ( N402K ) and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP - FKBP38 ( N402K ) was also monitored directly . Higher - magnification views of the boxed areas are shown in the right panels of each set . ( b ) MEFs stably expressing EGFP - FKBP38 ( N402K ) , mCherry - FKBP38 and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FLAG ( M2 ) . The fluorescence of EGFP - FKBP38 ( N402K ) and mCherry - FKBP38 was also monitored directly . ( c ) MEFs stably expressing FLAG - Parkin and either EGFP - FKBP38 or EGFP - FKBP38 ( N402K ) were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis . ( d ) Densitometric quantitation of EGFP fusion protein abundance in immunoblots similar to those in ( c ) . Data are expressed relative to the corresponding control value ( CCCP 0 h ) and are means ± s . d . , from three independent experiments . * * P0 . 01 ( Student ' s t test ) . ( e ) MEFs stably expressing EGFP - Bcl - 2 ( H235R ) and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP - Bcl - 2 ( H235R ) was also monitored directly . Higher - magnification views of the boxed areas are shown in the right panels of each set . ( g ) HeLa cells stably expressing FLAG - Parkin were transiently transfected with vectors for KikGR - FKBP38ΔN47 or KikGR - FKBP38ΔN47 ( N402K ) , exposed to UV , incubated in the absence or presence of CCCP ( 10 μM ) for 4 h , and then monitored for red and green KikGR fluorescence . Scale bar ( a , b , e , g ) , 10 μm .

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f7(a) MEFs stably expressing EGFP-FKBP38(N402K) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-FKBP38(N402K) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.(b) MEFs stably expressing EGFP-FKBP38(N402K), mCherry-FKBP38 and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FLAG (M2). The fluorescence of EGFP-FKBP38(N402K) and mCherry-FKBP38 was also monitored directly.(c) MEFs stably expressing FLAG-Parkin and either EGFP-FKBP38 or EGFP-FKBP38(N402K) were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis. (d) Densitometric quantitation of EGFP fusion protein abundance in immunoblots similar to those in (c). Data are expressed relative to the corresponding control value (CCCP 0 h) and are means±s.d., from three independent experiments. **P0.01 (Student's t test).(e) MEFs stably expressing EGFP-Bcl-2(H235R) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-Bcl-2(H235R) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.(g) HeLa cells stably expressing FLAG-Parkin were transiently transfected with vectors for KikGR-FKBP38ΔN47 or KikGR-FKBP38ΔN47(N402K), exposed to UV, incubated in the absence or presence of CCCP (10 μM) for 4 h, and then monitored for red and green KikGR fluorescence. Scale bar (a,b,e,g), 10 μm.


f8 ( a ) Wild - type ( Mfn2 + / + ) and Mfn2 knockout ( Mfn2 - / - ) MEFs stably expressing mCherry - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c . The fluorescence of mCherry - Parkin was also monitored directly . ( b ) MEFs as in a but expressing EGFP - Parkin were treated with CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis . The asterisk indicates a nonspecific band . ( c ) Wild - type MEFs stably expressing EGFP - Parkin and FLAG - VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) or nocodazole ( 10 μM ) for 6 h as indicated , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c , to FLAG and to FKBP38 . The fluorescence of EGFP - Parkin was also monitored directly . ( d ) Higher - magnification views of the boxed areas in ( c , e ) . Wild - type MEFs stably expressing FLAG - Parkin and EGFP - FKBP38 were incubated in the absence or presence of CCCP ( 30 μM ) for 10 h and then subjected to immuno - electron microscopic analysis with antibodies to GFP . Black arrowheads indicate the mitochondria . White arrowheads indicate small vesicle . ( f ) Wild - type ( Fkbp38 + / + ) and Fkbp38 knockout ( Fkbp38 − / − ) MEFs stably expressing FLAG - Parkin and either EGFP or EGFP - FKBP38 were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then stained with allophycocyanin ( APC ) - labelled annexin V and propidium iodide ( PI ) for flow cytometric analysis of the percentage of apoptotic cells ( annexin V + and PI + cells ) . Data are means ± s . d . , from 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

f8(a) Wild-type (Mfn2+/+) and Mfn2 knockout (Mfn2-/-) MEFs stably expressing mCherry-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c. The fluorescence of mCherry-Parkin was also monitored directly.(b) MEFs as in a but expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis. The asterisk indicates a nonspecific band.(c) Wild-type MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) or nocodazole (10 μM) for 6 h as indicated, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c, to FLAG and to FKBP38. The fluorescence of EGFP-Parkin was also monitored directly. (d) Higher-magnification views of the boxed areas in (c,e). Wild-type MEFs stably expressing FLAG-Parkin and EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 10 h and then subjected to immuno-electron microscopic analysis with antibodies to GFP. Black arrowheads indicate the mitochondria. White arrowheads indicate small vesicle.(f) Wild-type (Fkbp38+/+) and Fkbp38 knockout (Fkbp38−/−) MEFs stably expressing FLAG-Parkin and either EGFP or EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then stained with allophycocyanin (APC)-labelled annexin V and propidium iodide (PI) for flow cytometric analysis of the percentage of apoptotic cells (annexin V+ and PI+ cells). Data are means±s.d., from three independent experiments.


Figure 1 ( B - C ) Immunofluorescent ( IF ) staining of Pax7 and YY1 was performed on cryosections of myotome or limb bud from Ctrl or YY1cKO mice to confirm the ablation of YY1 in Pax7 expressing progenitors . Scale bar = 100 µm . ( D ) Representative images of Ctrl , Heterozygous ( Het ) and YY1cKO newborn ( P0 ) mice or embryos at E18 . 5 day . ( E ) Excised lungs from P0 Ctrl or YY1cKO pups were placed in a beaker for sinking test . The lung from YY1cKO pup sank whereas Ctrl floated . ( F ) P0 lungs were stained with Hematoxylin and Eosin ( H & E ) and representative images are shown . Scale bar = 100 µm . ( G ) Representative images of diaphragms ( Dps ) isolated from P0 Ctrl or YY1cKO littermates . ( H ) The above Dps were used for H & E or Immunohistochemistry ( IHC ) staining for MyHC . Representative images from 3 pairs of littermates are shown . Scale bar = 50 µm . The above harvested Dps were subjected to IF staining for MyHC together with Laminin . Scale bar = 50 µm . Quantifications of positively stained fibers per area are shown on the right ( n = 3 mice , each ) . The above harvested Dps were subjected to IF staining for Troponin T ( TnT ) together with Laminin . Scale bar = 50 µm . Quantifications of positively stained fibers per area are shown on the right ( n = 3 mice , each ) . ( K ) Representative images of H & E staining of hind limb muscles isolated from Ctrl or YY1cKO embryos at E18 . 5 day . Scale bar = 400 µm ( left ) or 50 µm ( right ) . The above harvested limb muscles were subjected to IF staining for MyHC together with Laminin . Scale bar = 400 µm ( left ) or 50 µm ( right ) . Quantifications of the numbers of MyHC + fibers per area are shown on the right ( n = 3 mice , each ) . The above harvested limb muscles were subjected to IF staining for TnT together with Laminin . Scale bar = 400 µm ( left ) or 50 µm ( right ) . Quantifications of the numbers of TnT + fibers per area are shown on the right ( n = 3 mice , each ) . The above harvested limb ( N ) muscles were subjected to IF staining for Pax7 and Laminin . Scale bar = 100 µm ( N ) Quantification of the numbers of Pax7 + muscle progenitor cells per area are shown on the right ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . The above harvested diaphragm ( O ) muscles were subjected to IF staining for Pax7 and Laminin . Scale bar = 50 µm ( O ) . Quantification of the numbers of Pax7 + muscle progenitor cells per area are shown on the right ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

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 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 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 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 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 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 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 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 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

Figure 1(B-C) Immunofluorescent (IF) staining of Pax7 and YY1 was performed on cryosections of myotome or limb bud from Ctrl or YY1cKO mice to confirm the ablation of YY1 in Pax7 expressing progenitors. Scale bar = 100 µm.(D) Representative images of Ctrl, Heterozygous (Het) and YY1cKO newborn (P0) mice or embryos at E18.5 day.(E) Excised lungs from P0 Ctrl or YY1cKO pups were placed in a beaker for sinking test. The lung from YY1cKO pup sank whereas Ctrl floated.(F) P0 lungs were stained with Hematoxylin and Eosin (H&E) and representative images are shown. Scale bar = 100 µm. (G) Representative images of diaphragms (Dps) isolated from P0 Ctrl or YY1cKO littermates.(H) The above Dps were used for H&E or Immunohistochemistry (IHC) staining for MyHC. Representative images from 3 pairs of littermates are shown. Scale bar = 50 µm.The above harvested Dps were subjected to IF staining for MyHC together with Laminin. Scale bar = 50 µm. Quantifications of positively stained fibers per area are shown on the right (n=3 mice, each).The above harvested Dps were subjected to IF staining for Troponin T (TnT) together with Laminin. Scale bar = 50 µm. Quantifications of positively stained fibers per area are shown on the right (n=3 mice, each).(K) Representative images of H&E staining of hind limb muscles isolated from Ctrl or YY1cKO embryos at E18.5 day. Scale bar = 400 µm (left) or 50 µm (right).The above harvested limb muscles were subjected to IF staining for MyHC together with Laminin. Scale bar = 400 µm (left) or 50 µm (right). Quantifications of the numbers of MyHC+ fibers per area are shown on the right (n=3 mice, each).The above harvested limb muscles were subjected to IF staining for TnT together with Laminin. Scale bar = 400 µm (left) or 50 µm (right). Quantifications of the numbers of TnT+ fibers per area are shown on the right (n=3 mice, each).The above harvested limb (N) muscles were subjected to IF staining for Pax7 and Laminin. Scale bar = 100 µm (N) Quantification of the numbers of Pax7+ muscle progenitor cells per area are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.The above harvested diaphragm (O) muscles were subjected to IF staining for Pax7 and Laminin. Scale bar = 50 µm (O). Quantification of the numbers of Pax7+ muscle progenitor cells per area are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01 ***P<0.001.


Figure 2 ( B ) SCs were FACS sorted 3 days after the last TM injection and cultured for 1 . 5 days ; RT - qPCR detection of YY1 mRNA shows the ablation in YY1iKO cells . ( C ) IF staining for Pax7 and YY1 on freshly isolated FISCs or ( D ) single myofibers from EDL muscles or ( E ) cryosections from TA muscles showing the deletion of YY1 protein from YY1iKO cells . Scale bar = 100 µm in ( C ) or 50 µm in ( D - E ) . ( F ) Representative FACS plots . About 100 , 000 cells from 2 - month - old Ctrl and YY1iKO mice were sorted by FACS 3 weeks post - TM injection . The percentage of SCs was shown . ( n = 3 mice , each ) . ( G ) H & E staining was performed on the injured TA muscles collected from the designated times post - CTX injection to visualize the degree of regeneration . Scale bar = 100 µm . ( H ) IF staining for eMyHC on the TA muscles 5 days post - CTX injury . Quantifications of the number of eMyHC + fibers are shown on the right ( n = 3 , each ) . Scale bar = 50 µm . ( I ) Left : representative images of TA muscles isolated from Ctrl or YY1iKO mice 60 days post - CTX injury . Right : Masson ' s Trichrome staining of the above muscles to visualize the degree of fibrosis . Scale bar = 100 µm . ( J ) Cross - section area ( CSA ) of individual myofibers in TA muscles 4 weeks after injury was measured . The distribution for CSA was calculated ( n = 3 mice ) . ( K ) Representative FACS plots showing the percentage of SCs sorted from TA muscles 3 days after CTX injury of Ctrl and YY1iKO mice . Immunostaining for Pax7 together with Laminin was performed on the TA muscles 3 days post - CTX injury . Scale bar = 100 µm . Quantifications of the numbers of Pax7 + SCs per area are shown on the right ( n = 3 mice , each ) . Immunostaining for MyoD together with Laminin was performed on the TA muscles 3 days post - CTX injury . Scale bar = 100 µm . Quantifications of the numbers of MyoD + SCs per area are shown on the right ( n = 3 mice , each ) . ( N ) IF staining for Pax7 and Laminin on TA muscles 4 weeks after CTX injury of Ctrl or YY1iKO mice . Quantifications of the numbers of Pax7 + SCs per 100 fibers are shown on the right ( n = 3 mice , each ) . Scale bar = 100 μm . ( P ) H & E staining was performed on TA muscles of Ctrl or YY1iKO mice 7 day after second CTX injury . Scale bar = 400 μm ( left ) or 100 μm ( right ) . ( Q ) Representative images of above TA muscles 7 days after second injury . ( R ) Quantifications of the weight above TA muscles from three pairs of littermate mice . ( S ) IF staining for eMyHC and Laminin on TA muscles 7 days after the second CTX injury of Ctrl or YY1iKO mice . Quantifications of the number of eMyHC + fibers per area are shown on the right ( n = 3 mice , each ) . Scale bar = 100 μm . ( T ) IF staining for Pax7 and Laminin on the above muscles and the quantifications of the number of Pax7 + SCs per area are shown on the right ( n = 3 mice , each ) . Scale bar = 100 μm . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 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 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 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 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 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 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 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 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 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 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 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

Figure 2(B) SCs were FACS sorted 3 days after the last TM injection and cultured for 1.5 days; RT-qPCR detection of YY1 mRNA shows the ablation in YY1iKO cells.(C) IF staining for Pax7 and YY1 on freshly isolated FISCs or (D) single myofibers from EDL muscles or (E) cryosections from TA muscles showing the deletion of YY1 protein from YY1iKO cells. Scale bar =100 µm in (C) or 50 µm in (D-E).(F) Representative FACS plots. About 100,000 cells from 2-month-old Ctrl and YY1iKO mice were sorted by FACS 3 weeks post-TM injection. The percentage of SCs was shown. (n=3 mice, each).(G) H&E staining was performed on the injured TA muscles collected from the designated times post-CTX injection to visualize the degree of regeneration. Scale bar = 100 µm.(H) IF staining for eMyHC on the TA muscles 5 days post-CTX injury. Quantifications of the number of eMyHC+ fibers are shown on the right (n=3, each). Scale bar = 50 µm.(I) Left: representative images of TA muscles isolated from Ctrl or YY1iKO mice 60 days post-CTX injury. Right: Masson's Trichrome staining of the above muscles to visualize the degree of fibrosis. Scale bar = 100 µm.(J) Cross-section area (CSA) of individual myofibers in TA muscles 4 weeks after injury was measured. The distribution for CSA was calculated (n=3 mice).(K) Representative FACS plots showing the percentage of SCs sorted from TA muscles 3 days after CTX injury of Ctrl and YY1iKO mice.Immunostaining for Pax7 together with Laminin was performed on the TA muscles 3 days post-CTX injury. Scale bar = 100 µm. Quantifications of the numbers of Pax7+ SCs per area are shown on the right (n=3 mice, each).Immunostaining for MyoD together with Laminin was performed on the TA muscles 3 days post-CTX injury. Scale bar = 100 µm. Quantifications of the numbers of MyoD+ SCs per area are shown on the right (n=3 mice, each).(N) IF staining for Pax7 and Laminin on TA muscles 4 weeks after CTX injury of Ctrl or YY1iKO mice. Quantifications of the numbers of Pax7+ SCs per 100 fibers are shown on the right (n=3 mice, each). Scale bar = 100 μm.(P) H&E staining was performed on TA muscles of Ctrl or YY1iKO mice 7 day after second CTX injury. Scale bar = 400 μm (left) or 100 μm (right).(Q) Representative images of above TA muscles 7 days after second injury.(R) Quantifications of the weight above TA muscles from three pairs of littermate mice.(S) IF staining for eMyHC and Laminin on TA muscles 7 days after the second CTX injury of Ctrl or YY1iKO mice. Quantifications of the number of eMyHC+ fibers per area are shown on the right (n=3 mice, each). Scale bar = 100 μm.(T) IF staining for Pax7 and Laminin on the above muscles and the quantifications of the number of Pax7+ SCs per area are shown on the right (n=3 mice, each). Scale bar = 100 μm. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.