D Accumulation of damaged lysosomes in affected p97 patient skeletal muscle . Patient biopsies immunostained for Gal3 and caveolin - 3 ( Cav3 ) . Quantification from 4 random 10X fields for each biopsy and means for controls and patients , respectively . Patients carry p97 mutations R155H ( # 1 - 3 ) or R93C for ( # 4 ) . Patients # 2 and # 3 are cousins . Scale bar , 100 µm .

D Accumulation of damaged lysosomes in affected p97 patient skeletal muscle. Patient biopsies immunostained for Gal3 and caveolin-3 (Cav3). Quantification from 4 random 10X fields for each biopsy and means for controls and patients, respectively. Patients carry p97 mutations R155H (#1-3) or R93C for (#4). Patients #2 and #3 are cousins. Scale bar, 100 µm.


A Candidate screen for p97 cofactors involved in autophagy of damaged lysosomes . HeLa cells were transfected with indicated siRNA oligos and LLOMe - treated for 1 h . Cells were chased for 12 h , fixed and stained for Gal3 . See Figure EV3 for depletion efficiency . B Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition . This threshold was chosen so that less than 1 % of untreated cells were considered positive . Data represent mean ± SEM of three independent experiments with ≥ 30 cells quantified per condition . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - sided Welch ' s t - tests ) .

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A Candidate screen for p97 cofactors involved in autophagy of damaged lysosomes. HeLa cells were transfected with indicated siRNA oligos and LLOMe-treated for 1 h. Cells were chased for 12 h, fixed and stained for Gal3. See Figure EV3 for depletion efficiency.B Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition. This threshold was chosen so that less than 1% of untreated cells were considered positive. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-sided Welch's t-tests).


C YOD1 , UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes . Cells expressing indicated tagged proteins were LLOMe - treated for 1 h or with vehicle alone ( control ) , fixed 2 h after washout , and stained for endogenous LAMP1 and , in the case of PLAA , with Alexa - 488 conjugated anti - HA antibodies . See Figure EV3B for quantification .

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C YOD1, UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes. Cells expressing indicated tagged proteins were LLOMe-treated for 1 h or with vehicle alone (control), fixed 2 h after washout, and stained for endogenous LAMP1 and, in the case of PLAA, with Alexa-488 conjugated anti-HA antibodies. See Figure EV3B for quantification.


D An ATP - stabilized complex of p97 containing both UBXD1 and PLAA . Stable HEK293 cell lines were doxycycline - induced to express p97 wild - type ( wt ) or the ATPase - mutant E578Q ( EQ ) at near endogenous level . Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies . Arrowheads indicate endogenous ( lower band ) and induced p97 ( upper band ) .

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D An ATP-stabilized complex of p97 containing both UBXD1 and PLAA. Stable HEK293 cell lines were doxycycline-induced to express p97 wild-type (wt) or the ATPase-mutant E578Q (EQ) at near endogenous level. Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies. Arrowheads indicate endogenous (lower band) and induced p97 (upper band).


E Stable HEK293 cells treated as in ( D ) and transiently expressing GFP - tagged YOD1 - wt or the YOD1 - C160S catalytic mutant ( CS ) were processed for co - immunoprecipitation with GFP nanobodies . Asterisk , unspecific band . Note that the YOD1 - CS mutant stabilized p97 binding and , in the p97 - EQ background , also associated with UBXD1 and PLAA .

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E Stable HEK293 cells treated as in (D) and transiently expressing GFP-tagged YOD1-wt or the YOD1-C160S catalytic mutant (CS) were processed for co-immunoprecipitation with GFP nanobodies. Asterisk, unspecific band. Note that the YOD1-CS mutant stabilized p97 binding and, in the p97-EQ background, also associated with UBXD1 and PLAA.


F Control ( IgG ) and UBXD1co - immunoprecipitation with indicated associated proteins from HeLa cells expressing GFP - YOD1 - CS after formaldehyde cross - linking . Ufd1 served as a negative control . Scale bars , 10 µm .

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F Control (IgG) and UBXD1co-immunoprecipitation with indicated associated proteins from HeLa cells expressing GFP-YOD1-CS after formaldehyde cross-linking. Ufd1 served as a negative control. Scale bars, 10 µm.


A K63 - and K48 - ubiquitination of damaged lysosomes . Untransfected HeLa cells , or cells expressing mCherry - Gal3 ( Ch - Gal3 ) were LLOMe - treated for 1 h . 2 h after washout , cells were stained with antibodies specific for K63 or K48 chains , and LAMP1 or p62 as indicated . Percentage of K63 - or K48 - ubiquitinated LAMP1 vesicles and percentage of p62 - positive K63 or K48 vesicles were quantified from three independent experiments ( mean ± SD , Student ' s unpaired t - test for LAMP1 ) . * = p < 0 . 05 .

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A K63- and K48-ubiquitination of damaged lysosomes. Untransfected HeLa cells, or cells expressing mCherry-Gal3 (Ch-Gal3) were LLOMe-treated for 1 h. 2 h after washout, cells were stained with antibodies specific for K63 or K48 chains, and LAMP1 or p62 as indicated. Percentage of K63- or K48-ubiquitinated LAMP1 vesicles and percentage of p62-positive K63 or K48 vesicles were quantified from three independent experiments (mean ± SD, Student's unpaired t-test for LAMP1). * = p < 0.05.


B Colocalization of K63 - and K48 - linked ubiquitin conjugates on individual lysosomes . Cells expressing GFP - TAB2 - NZF ( K63 - sensor ) were treated as in ( A ) and stained for K48 chains and LAMP1 . Note that the apparent nuclear localization of the sensor is a consequence of the extraction procedure ( van Wijk et al . , 2012 ) .

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B Colocalization of K63- and K48-linked ubiquitin conjugates on individual lysosomes. Cells expressing GFP-TAB2-NZF (K63-sensor) were treated as in (A) and stained for K48 chains and LAMP1. Note that the apparent nuclear localization of the sensor is a consequence of the extraction procedure (van Wijk et al., 2012).


C Cells expressing GFP - p97 - EQ were treated as in ( A ) and stained with antibodies as indicated . D Quantification of the total number of K63 - or K48 - ubiquitinated vesicles per cell , and number of p97 - EQ positive K63 - or K48 - ubiquitinated vesicles ( mean ± SD ) . E Percentage of GFP - p97 - EQ vesicles positive for K63 or K48 chains was determined ( mean ± SD ) .

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C Cells expressing GFP-p97-EQ were treated as in (A) and stained with antibodies as indicated.D Quantification of the total number of K63- or K48-ubiquitinated vesicles per cell, and number of p97-EQ positive K63- or K48-ubiquitinated vesicles (mean ± SD).E Percentage of GFP-p97-EQ vesicles positive for K63 or K48 chains was determined (mean ± SD).


F ELDR components target K48 - conjugates on lysosomes . Detection of GFP - YOD1 - CS or UBXD1 - GFP alone , or co - transfected with PLAA - HA and GFP - p97 - EQ , 2 h after LLOMe washout and co - stained with indicated antibodies .

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F ELDR components target K48-conjugates on lysosomes. Detection of GFP-YOD1-CS or UBXD1-GFP alone, or co-transfected with PLAA-HA and GFP-p97-EQ, 2 h after LLOMe washout and co-stained with indicated antibodies.


A Time course of K63 - and K48 - ubiquitination . Control - depleted cells were fixed at indicated time points after LLOMe washout and stained with chain - specific antibodies and LAMP1 . Automated quantification of LAMP1 vesicles positive for K63 or K48 chains . Data represent mean ± SD of three independent experiments . B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes . Experiment and quantification as in ( A ) in cells treated with p97 or YOD1 siRNA oligos .

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A Time course of K63- and K48-ubiquitination. Control-depleted cells were fixed at indicated time points after LLOMe washout and stained with chain-specific antibodies and LAMP1. Automated quantification of LAMP1 vesicles positive for K63 or K48 chains. Data represent mean ± SD of three independent experiments.B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes. Experiment and quantification as in (A) in cells treated with p97 or YOD1 siRNA oligos.


C The p97 - EQ mutant stabilizes K48 - conjugates . Lysosome damage was induced as in ( A ) in cells transfected with GFP - tagged p97 - wt or p97 - EQ . Cells were chased after washout for 12 h and stained for K48 chains . D Quantification of K48 - positive lysosomes 2 h and 12 h after damage in cells treated as in ( A ) . Data represent mean ± SD of three independent experiments .

C The p97-EQ mutant stabilizes K48-conjugates. Lysosome damage was induced as in (A) in cells transfected with GFP-tagged p97-wt or p97-EQ. Cells were chased after washout for 12 h and stained for K48 chains.D Quantification of K48-positive lysosomes 2 h and 12 h after damage in cells treated as in (A). Data represent mean ± SD of three independent experiments.


E The catalytically - inactive mutant of YOD1 stabilizes K48 - conjugates . Cells transfected with GFP - tagged YOD1 - wt or the YOD1 - CS were treated and stained as in ( C ) . F Quantification as in ( D ) of cells treated as in ( E ) . * * = p < 0 . 01 * * * = p < 0 . 001 ( Student ' s unpaired t - test ) . ns , not significant . Scale bars , 10 µm .

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E The catalytically-inactive mutant of YOD1 stabilizes K48-conjugates. Cells transfected with GFP-tagged YOD1-wt or the YOD1-CS were treated and stained as in (C).F Quantification as in (D) of cells treated as in (E). ** = p < 0.01 *** = p < 0.001 (Student's unpaired t-test). ns, not significant. Scale bars, 10 µm.


A YOD1 can link p97 to ubiquitin . Ubiquitin - GST ( Ub - GST ) or GST alone was incubated with the indicated purified proteins and isolated . Co - isolated proteins ( pulldown ) were detected in SDS - gels . Note binding of p97 and YOD1 to Ub - GST only in combination .

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A YOD1 can link p97 to ubiquitin. Ubiquitin-GST (Ub-GST) or GST alone was incubated with the indicated purified proteins and isolated. Co-isolated proteins (pulldown) were detected in SDS-gels. Note binding of p97 and YOD1 to Ub-GST only in combination.


B The S2 site of YOD1 is critical for ubiquitin binding . Experiments as in ( A ) with indicated combinations of p97 and YOD1 - wt or the ubiquitin - binding mutant , MutS2 .

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B The S2 site of YOD1 is critical for ubiquitin binding. Experiments as in (A) with indicated combinations of p97 and YOD1-wt or the ubiquitin-binding mutant, MutS2.


C Reconstitution of a complex of ELDR components on ubiquitin . Experiments as in ( A ) with combinations of p97 - wt or p97 - EQ , YOD1 , PLAA and UBXD1 as indicated . Note that PLAA associates with p97 and YOD1 on ubiquitin only in the case of p97 - EQ .

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C Reconstitution of a complex of ELDR components on ubiquitin. Experiments as in (A) with combinations of p97-wt or p97-EQ, YOD1, PLAA and UBXD1 as indicated. Note that PLAA associates with p97 and YOD1 on ubiquitin only in the case of p97-EQ.


D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site . LLOMe - treated cells expressing GFP - YOD1 - CS or the double mutant GFP - YOD1 - CS - MutS2 were chased for 2 h and stained for K48 chains and LAMP1 . Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined .

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D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site. LLOMe-treated cells expressing GFP-YOD1-CS or the double mutant GFP-YOD1-CS-MutS2 were chased for 2 h and stained for K48 chains and LAMP1. Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined.


E The S2 site is essential for K48 - conjugate removal . GFP - YOD1 - wt or GFP - YOD1 - MutS2 overexpressing cells treated as in ( D ) were stained for K48 chains . Quantification of the number of K48 - positive vesicles per cell . Data represent mean ± SD of three independent experiments . * * = p < 0 . 01 ( Student ' s unpaired t - test ) . Scale bars , 10 µm .

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E The S2 site is essential for K48-conjugate removal. GFP-YOD1-wt or GFP-YOD1-MutS2 overexpressing cells treated as in (D) were stained for K48 chains. Quantification of the number of K48-positive vesicles per cell. Data represent mean ± SD of three independent experiments. ** = p < 0.01 (Student's unpaired t-test). Scale bars, 10 µm.


A Largely mutually exclusive localization of LC3 and K48 chains . GFP - LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains . Correlation coefficients were calculated for colocalization of LC3 with K63 or K48 . The whiskers represent 10th and 90th percentile . Points below and above the whiskers are drawn as individual dots . The single line indicates the median . * * * = p < 0 . 001 ( Mann - Whitney - U test ) .

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A Largely mutually exclusive localization of LC3 and K48 chains. GFP-LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains. Correlation coefficients were calculated for colocalization of LC3 with K63 or K48. The whiskers represent 10th and 90th percentile. Points below and above the whiskers are drawn as individual dots. The single line indicates the median. *** = p < 0.001 (Mann-Whitney-U test).


B Lipofectamine - coated latex beads damage lysosomes and induce their K48 - ubiquitination . Cells expressing mCherry - Gal3 were incubated with Lipofectamine - coated latex beads for 3 h and stained with K48 chain - specific antibodies .

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B Lipofectamine-coated latex beads damage lysosomes and induce their K48-ubiquitination. Cells expressing mCherry-Gal3 were incubated with Lipofectamine-coated latex beads for 3 h and stained with K48 chain-specific antibodies.


C YOD1 association and dissociation precedes LC3 recruitment . Live cell video microscopy stills of cells co - expressing mCherry - YOD1 - wt and GFP - LC3 after treatment with Lipofectamine - coated latex beads for 30 min at 2 min image intervals .

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C YOD1 association and dissociation precedes LC3 recruitment. Live cell video microscopy stills of cells co-expressing mCherry-YOD1-wt and GFP-LC3 after treatment with Lipofectamine-coated latex beads for 30 min at 2 min image intervals.


D Stabilization of K48 - conjugates on lysosomes impairs recruitment of LC3 . Cells expressing GFP - YOD1 - CS were treated as in ( A ) and stained for LAMP1 and p62 or LC3 . Percentage of GFP - YOD1 vesicles positive for p62 or LC3 was determined ( mean ± SD ) .

D Stabilization of K48-conjugates on lysosomes impairs recruitment of LC3. Cells expressing GFP-YOD1-CS were treated as in (A) and stained for LAMP1 and p62 or LC3. Percentage of GFP-YOD1 vesicles positive for p62 or LC3 was determined (mean ± SD).


E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation . Cells were transfected with control ( Ctrl ) , p97 or YOD1 siRNA oligos , treated with LLOMe , fixed after 12 h chase and stained for endogenous LC3 and K48 chains . Quantification of the number of K48 - positive vesicles per cell and number of LC3 - positive K48 vesicles per cell was determined . Data represent mean ± SD of three independent experiments . Scale bars , 10 µm .

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E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation. Cells were transfected with control (Ctrl), p97 or YOD1 siRNA oligos, treated with LLOMe, fixed after 12 h chase and stained for endogenous LC3 and K48 chains. Quantification of the number of K48-positive vesicles per cell and number of LC3-positive K48 vesicles per cell was determined. Data represent mean ± SD of three independent experiments. Scale bars, 10 µm.


b . Heatmap depicting mean expression of the top 20 upregulated genes of fibroblast isolated from wound - induced tumours ( Pap ) compared to fibroblasts from wild - type ( WT ) and inflamed ( InvEE ) back skin ( n = 3 per condition ) . PRSS35 is indicated in red as a top upregulated gene in CAFs .

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b. Heatmap depicting mean expression of the top 20 upregulated genes of fibroblast isolated from wound-induced tumours (Pap) compared to fibroblasts from wild-type (WT) and inflamed (InvEE) back skin (n=3 per condition). PRSS35 is indicated in red as a top upregulated gene in CAFs.


a . Immunofluorescent images of skin sections from PDGFRα - eGFP reporter mice with normal ( WT , n = 4 ) , inflamed ( InvEE , n = 4 ) , wounded ( d14 post - wounding ; wound , n = 4 ) , or tumour - associated ( Papilloma , Pap d17 and d30 post - wounding , n = 4 per condition ) skin . Nuclei were stained with Dapi ( 4 ' , 6 - diamidino - 2 - phenylindole ; blue ) . Dotted line represents epidermal - dermal boundary . Scale bars : 50 μm . b . Quantification of the number of fibroblasts present in different skin conditions ( n = 4 mice per condition ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; One - way ANOVA testing ) . Data represent means of multiple microscopic fields ± S . E . M .

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a. Immunofluorescent images of skin sections from PDGFRα-eGFP reporter mice with normal (WT, n=4), inflamed (InvEE, n=4), wounded (d14 post-wounding; wound, n=4), or tumour-associated (Papilloma, Pap d17 and d30 post-wounding, n=4 per condition) skin. Nuclei were stained with Dapi (4', 6-diamidino- 2- phenylindole; blue). Dotted line represents epidermal-dermal boundary. Scale bars: 50 μm. b. Quantification of the number of fibroblasts present in different skin conditions (n=4 mice per condition; ** p<0.01; *** p<0.001; **** p<0.0001; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M.


c . Herovici staining of sections of normal ( WT ) , inflamed ( InvEE ) , wound , or tumour - associated ( Papilloma ) skin . Scale bars : 100 μm .

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c. Herovici staining of sections of normal (WT), inflamed (InvEE), wound, or tumour-associated (Papilloma) skin. Scale bars: 100 μm.


c . Relative mRNA levels of PRSS35 in lysates from normal skin ( WT , n = 4 mice ) , inflamed skin ( InvEE , n = 4 mice ) , wounds ( d14pw , n = 4 mice ) and wound - induced papillomas ( Pap , n = 5 mice ) . ( * p = 0 . 0159 ; Mann - Whitney test ) . Data represent means of three technical replicates ± S . E . M .

c. Relative mRNA levels of PRSS35 in lysates from normal skin (WT, n=4 mice), inflamed skin (InvEE, n=4 mice), wounds (d14pw, n=4 mice) and wound-induced papillomas (Pap, n=5 mice). (* p=0.0159; Mann-Whitney test). Data represent means of three technical replicates ± S.E.M.


f . H & E - stained skin sections of WT and PRSS35 - / - wounds at different days post - wounding . Scale bars : 100 µm .

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f. H&E-stained skin sections of WT and PRSS35-/- wounds at different days post-wounding. Scale bars: 100 µm.


c , d . Incidence of papilloma formation ( ns : non - significant ; Gehan - Breslow - Wilcoxon test ) ( c ) and number of tumours per mouse ( d ) in wild - type ( WT , n = 17 ) and PRSS35 - / - ( n = 22 ) mice treated with DMBA - TPA ( * p = 0 . 0171 ; Wilcoxon matched - paired rank test ) . Data represent means ± S . E . M .

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c, d. Incidence of papilloma formation (ns: non-significant; Gehan-Breslow-Wilcoxon test) (c) and number of tumours per mouse (d) in wild-type (WT, n=17) and PRSS35-/- (n=22) mice treated with DMBA-TPA (*p=0.0171; Wilcoxon matched-paired rank test). Data represent means ± S.E.M.


b . Relative mRNA expression levels of the indicated ECM genes in primary fibroblast cultures ( col1a1 : collagen type 1 alpha 1 ; αSMA , alpha smooth muscle actin ; Cyr61 : Cysteine - rich angiogenic inducer 61 ( = CCN1 ) ) in primary fibroblast cultures isolated from WT and PRSS35 - / - mice , untreated or after 24 hours stimulation with 10 ng / mL TGFβ1 ( n ≥ 7 biological replicates per condition ) ( * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ; Two - way ANOVA with multiple comparisons ) . Data represent means ± S . E . M . c Relative mRNA expression levels of the indicated cytokines / growth factors in primary fibroblast cultures ( n ≥ 8 per condition ) ( VEGF - α , vascular endothelial growth factor - alpha ; FGF2 : basic fibroblast growth factor ; FGF7 : keratinocyte growth factor ) ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ; Two - way ANOVA with multiple comparisons ) . Data represent means ± S . E . M .

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b. Relative mRNA expression levels of the indicated ECM genes in primary fibroblast cultures (col1a1: collagen type 1 alpha 1; αSMA, alpha smooth muscle actin; Cyr61: Cysteine-rich angiogenic inducer 61 (=CCN1)) in primary fibroblast cultures isolated from WT and PRSS35-/- mice, untreated or after 24 hours stimulation with 10 ng/mL TGFβ1 (n≥7 biological replicates per condition) (** p<0.01, *** p<0.001, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M. c Relative mRNA expression levels of the indicated cytokines/growth factors in primary fibroblast cultures (n≥8 per condition) (VEGF-α, vascular endothelial growth factor-alpha; FGF2: basic fibroblast growth factor; FGF7: keratinocyte growth factor) (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M.


d . Picrosirius red staining of normal skin , day 14 post - wounding ( d14pw ) skin or wound - induced papillomas from WT and PRSS35 KO mice . Red colouring indicates thick fibres , green colouring indicates thin fibres . Scale bars : 200 µm . e . Quantification of picrosirius red staining as total collagen density ( upper panel ) and ratio of red versus green picrosirius staining ( lower panel ) . Using QuPath Bioimage analysis software pixels colouring red or green were determined per area . ( n ≥ 4 technical replicates per condition ) ( * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . ; Two - way ANOVA with multiple comparisons ) . Data represent means ± S . E . M .

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d. Picrosirius red staining of normal skin, day 14 post-wounding (d14pw) skin or wound-induced papillomas from WT and PRSS35 KO mice. Red colouring indicates thick fibres, green colouring indicates thin fibres. Scale bars: 200 µm. e. Quantification of picrosirius red staining as total collagen density (upper panel) and ratio of red versus green picrosirius staining (lower panel). Using QuPath Bioimage analysis software pixels colouring red or green were determined per area. (n≥4 technical replicates per condition) (* p<0.05, ** p<0.01, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M.


a . Human skin sections were stained with a PRSS35 - specific antibody ( red ) and counterstained with Dapi ( n = 5 per condition ) . Boxed areas are magnifications as shown in the lower panel and dotted line represents the epidermal - dermal boundary . Scale bars : 100 μm . b . Quantification of PRSS35 positive signal by Volocity analysis ( n ≥ 4 per condition ; * * p < 0 . 01 ; One - way ANOVA testing ) . Data represent means of multiple microscopic fields ± S . E . M .

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a. Human skin sections were stained with a PRSS35-specific antibody (red) and counterstained with Dapi (n=5 per condition). Boxed areas are magnifications as shown in the lower panel and dotted line represents the epidermal-dermal boundary. Scale bars: 100 μm. b. Quantification of PRSS35 positive signal by Volocity analysis (n≥4 per condition; ** p<0.01; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M.


( C ) Cleavage of supercoiled ( SC ) plasmid DNA substrates using purified Cas12l nuclease and respective sgRNA variants . Efficient full length linearization ( FLL ) of the substrate resulting from a complete double - strand break was only observed when using sgRNAs bearing the 5 ' most end of the tracrRNA ( variants 1 and 3 ) . Moreover , the terminator - like Hairpin 3 is not required for target cleavage ( variant 1 ) . OC - open circular ; FLL - full length linear ; SC - supercoiled .

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(C) Cleavage of supercoiled (SC) plasmid DNA substrates using purified Cas12l nuclease and respective sgRNA variants. Efficient full length linearization (FLL) of the substrate resulting from a complete double-strand break was only observed when using sgRNAs bearing the 5' most end of the tracrRNA (variants 1 and 3). Moreover, the terminator-like Hairpin 3 is not required for target cleavage (variant 1). OC - open circular; FLL - full length linear; SC- supercoiled.


( B ) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l . wt - wildtype .

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(B) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l. wt - wildtype.


( C ) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition . The molar ratio of RNP to substrate was kept low ( 5 : 1 ) to increase reaction stringency . Asp2Cas12l predominantly recognizes a 5 ' - CCY - 3 ' PAM and Asp3Cas12l a 5 ' - CCB - 3 ' PAM . N = 3 . Data information : In ( C ) , individual data points are plotted , where n = 4 replicates from independent experiments ( Asp2Cas12l ) and n = 3 replicates from independent experiments ( Asp3Cas12l ) . The data points were fitted to a single exponential association curve ( solid lines ) .

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(C) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition. The molar ratio of RNP to substrate was kept low (5:1) to increase reaction stringency. Asp2Cas12l predominantly recognizes a 5'-CCY-3' PAM and Asp3Cas12l a 5'-CCB-3' PAM. N=3. Data information: In (C), individual data points are plotted, where n = 4 replicates from independent experiments (Asp2Cas12l) and n = 3 replicates from independent experiments (Asp3Cas12l). The data points were fitted to a single exponential association curve (solid lines).


( A ) Asp2 and Asp3Cas12l RNP complexes degrade M13 ssDNA in the presence of single - stranded ( ss ) DNA or dsDNA ( including PAM ) activators with target sequences complementary to the gRNA spacer . NS activator - non specific activator ( ssDNA oligonucleotide or dsDNA duplex ) with no sequence complementarity to the spacer of the gRNA .

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(A) Asp2 and Asp3Cas12l RNP complexes degrade M13 ssDNA in the presence of single-stranded (ss) DNA or dsDNA (including PAM) activators with target sequences complementary to the gRNA spacer. NS activator - non specific activator (ssDNA oligonucleotide or dsDNA duplex) with no sequence complementarity to the spacer of the gRNA.


( B ) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched fluorescent ssDNA or ssRNA probes . Background - subtracted traces from fluorescent reporter assays with favored ssDNA ( 5 ' - CCCCCCCC - 3 ' ) or ssRNA ( 5 ' - CCCCCCCC - 3 ' ) probes . RFU - relative fluorescence units . ( C ) Background - subtracted traces of fluorescent reporter cleavage using ssDNA or ssRNA probes . ( D ) Rates of trans - degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets . Collateral ssDNAse activity is about 3 - fold higher than the rate of ssRNA degradation . Fluorescence intensities in B - C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters . Data information : In ( B - D ) , individual data points from n = 3 replicates from independent experiments are plotted . In ( D ) , bars represent the mean .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched fluorescent ssDNA or ssRNA probes. Background-subtracted traces from fluorescent reporter assays with favored ssDNA (5'-CCCCCCCC-3') or ssRNA (5'-CCCCCCCC-3') probes. RFU - relative fluorescence units. (C) Background-subtracted traces of fluorescent reporter cleavage using ssDNA or ssRNA probes. (D) Rates of trans-degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets. Collateral ssDNAse activity is about 3-fold higher than the rate of ssRNA degradation. Fluorescence intensities in B-C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters. Data information: In (B-D), individual data points from n = 3 replicates from independent experiments are plotted. In (D), bars represent the mean.


( A ) Representative images of WT , Mfn1 KO and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria - targeted RFP ( mt - RFP ) . Scale bar : 5 μm .

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(A) Representative images of WT, Mfn1 KO and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria-targeted RFP (mt-RFP). Scale bar: 5 μm.


( A ) Representative images of WT and Mfn2 KO MEFs co - transfected with plasmids encoding mitochondria - targeted RFP and ER - targeted GFP . Scale bar : 5 μm .

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(A) Representative images of WT and Mfn2 KO MEFs co-transfected with plasmids encoding mitochondria-targeted RFP and ER-targeted GFP. Scale bar: 5 μm.


( B ) After 48 h , the cells were fixed and the degree of colocalization between ER and mitochondria based on red and green fluorescent pixel overlap was analyzed using Mander ' s coefficient ( n = 30 cells analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . * p < 0 . 05 , two - tailed Student ' s t - test .

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(B) After 48 h, the cells were fixed and the degree of colocalization between ER and mitochondria based on red and green fluorescent pixel overlap was analyzed using Mander's coefficient (n= 30 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test.


I - K ) Mitochondrial Ca2 + was determined in WT and Mfn2 KO MEFs loaded with Rhod2 . The effect of ( I ) caffeine - induced ( 20 mM ) , ( J ) IP3R activation with ATP - induced ( 100 μM ) and ( K ) histamine - induced ( 100 μM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 30 cells from 3 independent experiments ) . Data are presented as mean ± SEM .

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I-K) Mitochondrial Ca2+ was determined in WT and Mfn2 KO MEFs loaded with Rhod2. The effect of (I) caffeine-induced (20 mM), (J) IP3R activation with ATP-induced (100 μM) and (K) histamine-induced (100 μM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.


WT and Mfn2 KO cells were transfected with plasmids encoding : GFP and the indicated combination of non - targeting siRNA ( siCT ) , siRNA targeting MCU ( siMCU ) , ChiMERA or control ( indicated as WT or Mfn2 alone ) plasmids . ( R ) MMP was analyzed ( n = 90 cells analyzed in 3 independent experiments ) .

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WT and Mfn2 KO cells were transfected with plasmids encoding: GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (R) MMP was analyzed (n= 90 cells analyzed in 3 independent experiments).


WT and Mfn2 KO cells were transfected with plasmids encoding GFP and the indicated combination of non - targeting siRNA ( siCT ) , siRNA targeting MCU ( siMCU ) , ChiMERA or control ( indicated as WT or Mfn2 alone ) plasmids . ( S ) ATP levels of GFP + cells was analyzed after 24 h treatment with 2DG ( 10 mM ) ( n = 3 independent experiments ) .

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WT and Mfn2 KO cells were transfected with plasmids encoding GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (S) ATP levels of GFP+ cells was analyzed after 24 h treatment with 2DG (10 mM) (n= 3 independent experiments).


WT and Mfn2 KO MEFs were co - transfected with plasmids encoding : mt - RFP and ER - Mfn2 or control ( CT ) plasmid . After 48 h , the cells were fixed . ( C ) Representative images . Scale bar = 5 μm .

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WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: mt-RFP and ER-Mfn2 or control (CT) plasmid. After 48 h, the cells were fixed. (C) Representative images. Scale bar= 5 μm.


WT and Mfn2 KO MEFs were co - transfected with plasmids encoding : J - N ) GFP and ER - Mfn2 or control ( CT ) plasmid . ( J ) MMP was determined . The values were normalized to surrounding untransfected cells . ( n = 90 cells analyzed in 3 independent experiments ) . ( K ) After 24 h , the transfected cells were sorted and plated for another 24 h , after which cells were treated with 2 - DG ( 10 mM ) for 6 h , and ATP levels were measured ( n = 3 independent experiments ) . ( L ) Oxygen consumption was measured and ( M ) RCR and ( N ) SRC was calculated ( n = 4 independent experiments ) . Data are presented as mean ± SEM . Data information : * p < 0 . 05 , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: J-N) GFP and ER-Mfn2 or control (CT) plasmid. (J) MMP was determined. The values were normalized to surrounding untransfected cells. (n= 90 cells analyzed in 3 independent experiments). (K) After 24 h, the transfected cells were sorted and plated for another 24 h, after which cells were treated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n= 3 independent experiments). (L) Oxygen consumption was measured and (M) RCR and (N) SRC was calculated (n= 4 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT and Mfn1 / 2 double KO were transfected with : A ) mt - RFP , ER - GFP and plasmids expressing the indicated proteins . Mitochondria - ER colocalization was analyzed using Mander ' s coefficient ( n = 15 cells analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . B ) GFP and ERMITO - RLuc and indicated expression vectors . After 24 h , the transfected cells were sorted and plated for another 24 h , after which RLuc was analyzed ( n = 3 independent experiments ) . Data are presented as mean ± SEM . C ) Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity .

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WT and Mfn1/2 double KO were transfected with: A) mt-RFP, ER-GFP and plasmids expressing the indicated proteins. Mitochondria-ER colocalization was analyzed using Mander's coefficient (n= 15 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. B) GFP and ERMITO-RLuc and indicated expression vectors. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed (n= 3 independent experiments). Data are presented as mean ± SEM. C) Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT and Mfn1 / 2 double KO were transfected with : C ) GFP and the indicated plasmids . After 48 h , mitochondrial Ca2 + was determined ( n = 30 cells from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity .

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WT and Mfn1/2 double KO were transfected with: C) GFP and the indicated plasmids. After 48 h, mitochondrial Ca2+ was determined (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT and Mfn1 / 2 double KO were transfected with : D ) LAR - GECO1 and the indicated plasmids . After 48 h ER Ca2 + was released with caffeine ( 20 mM ) as indicated ( n = 25 - 27 cells from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity .

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WT and Mfn1/2 double KO were transfected with: D) LAR-GECO1 and the indicated plasmids. After 48 h ER Ca2+ was released with caffeine (20 mM) as indicated (n= 25-27 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT and Mfn1 / 2 double KO were transfected with : E ) GFP and the indicated plasmid . After 48 h , the effect of caffeine - induced ( 20 mM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 25 cells from 3 independent experiments ) . Data are presented as mean ± SEM .

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WT and Mfn1/2 double KO were transfected with: E) GFP and the indicated plasmid. After 48 h, the effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 25 cells from 3 independent experiments). Data are presented as mean ± SEM.


B ) Representative Western blots of the indicated proteins during the neuronal maturation in vitro ( n = 3 independent experiments ) . C ) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 ( n = 3 independent experiments ) . D

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B) Representative Western blots of the indicated proteins during the neuronal maturation in vitro (n= 3 independent experiments). C) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 (n= 3 independent experiments). D


D ) Primary neurons of tamoxifen - inducible Mfn2 KO mice were transfected at DIV3 with ER - GFP , mt - RFP plus the indicated plasmids . Tamoxifen - treated ( for 72 h ) and vehicle - treated neurons ( WT ; red dashed line ) were fixed and mitochondria - ER co - localization was analyzed using Mander ' s coefficient ( n = 15 neurons analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc test . Scale bar : 500 μm .

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D) Primary neurons of tamoxifen-inducible Mfn2 KO mice were transfected at DIV3 with ER-GFP, mt-RFP plus the indicated plasmids. Tamoxifen-treated (for 72 h) and vehicle-treated neurons (WT; red dashed line) were fixed and mitochondria-ER co-localization was analyzed using Mander's coefficient (n= 15 neurons analyzed in 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm.


( F ) neurite length measurement and representative tracings ( G ) of primary cortical neurons of WT and Mfn2 KO mice co - transfected at DIV4 with GFP and the indicated plasmids . After 72 h , the neurons were fixed , immunofluorescence with anti - GFP antibodies was performed and neurite length was measured ( n = 30 neurons from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc test . Scale bar : 500 μm . E

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(F) neurite length measurement and representative tracings (G) of primary cortical neurons of WT and Mfn2 KO mice co-transfected at DIV4 with GFP and the indicated plasmids. After 72 h, the neurons were fixed, immunofluorescence with anti-GFP antibodies was performed and neurite length was measured (n= 30 neurons from 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm. E


A . Immunostaining of P5 cerebellum with indicated antibodies . ( A ) low magnification . ( A ' - A ' ' ' ' ) high magnification . The solid line indicates the pial edge of the EGL . The dashed line demarcates the bottom of the KI67 - stained region . The outer EGL corresponds to the area between the solid and dashed lines , which contain GCPs .

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A. Immunostaining of P5 cerebellum with indicated antibodies. (A) low magnification. (A'-A'''') high magnification. The solid line indicates the pial edge of the EGL. The dashed line demarcates the bottom of the KI67-stained region. The outer EGL corresponds to the area between the solid and dashed lines, which contain GCPs.


Atoh1 KD analysis . ( G , H ) Atoh1 shRNA was introduced to P5 mice EGL together with H2B - EGFP plasmid and immunostaining for NEUROD1 ( Cyan ) and KI67 ( magenta ) was performed 24 hrs later . Nuclei of electroporated cells were labeled with GFP ( green ) . Images of Control ( G ) and Atoh1 KD ( H ) . Arrows indicate the cells of interest where GFP signals are co - localized with NEUROD1 and KI67 .

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Atoh1 KD analysis. (G, H) Atoh1 shRNA was introduced to P5 mice EGL together with H2B-EGFP plasmid and immunostaining for NEUROD1 (Cyan) and KI67 (magenta) was performed 24 hrs later. Nuclei of electroporated cells were labeled with GFP (green). Images of Control (G) and Atoh1 KD (H). Arrows indicate the cells of interest where GFP signals are co-localized with NEUROD1 and KI67.


( I ) Proportions of NEUROD1 + , KI67 + cells ( ND + GCPs ) among the electroporated GCPs ( GFP + , KI67 + cells ) were increased in the Atoh1 KD mice . ( J ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Student T - test was performed between Control ( N = 4 ) and Atoh1 shRNA ( sh # 1 : N = 3 , sh # 2 : N = 3 ) . Bars and error bars are representing as Mean ± SEM . * p < 0 . 05 , N . S . : Not Significant . Number of cells : 98 , 71 , 81 and 60 cells from N = 4 mice ( sclamble ) , 127 , 93 and 51 cells from N = 3 mice ( sh # 1 ) , 75 , 111 , 38 cells from N = 3 mice ( sh # 2 ) .

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

(I) Proportions of NEUROD1+, KI67+ cells (ND+GCPs) among the electroporated GCPs (GFP+, KI67+ cells) were increased in the Atoh1 KD mice. (J) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=4) and Atoh1 shRNA (sh#1: N=3, sh#2: N=3). Bars and error bars are representing as Mean ± SEM. *p < 0.05, N.S.: Not Significant. Number of cells: 98,71,81 and 60 cells from N=4 mice (sclamble), 127,93 and 51 cells from N=3 mice (sh#1), 75,111,38 cells from N=3 mice (sh#2).


B . Immunostaining of P5 cerebellum with indicated antibodies . Boxplots of the intensity of CCND1 immuno - signal ( B ' ' ' ' , normalized to the mean intensity of AT + GCPs ) . We measured the intensities of CCND1 in ATOH1 + / NEUROD1 - cells and NEUROD1 + / ATOH1 - cells . ATOH1 + / NEUROD1 + cells were not included in this quantification . These boxplots are showing the median ( central band ) , 25 % / 75 % quartiles ( boxes ) and the ranges ( lower whiskers represent the regions of lower box - 1 . 5 * IQR ( inter - quartile range ) , upper whisker represent the regions of upper box - 1 . 5 * IQR ) . Wilcoxon test were performed between AT + GCPs and ND + GCPs . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunostaining of P5 cerebellum with indicated antibodies. Boxplots of the intensity of CCND1 immuno-signal (B'''', normalized to the mean intensity of AT+GCPs). We measured the intensities of CCND1 in ATOH1+/NEUROD1- cells and NEUROD1+/ ATOH1- cells. ATOH1+/ NEUROD1+ cells were not included in this quantification. These boxplots are showing the median (central band), 25% / 75% quartiles (boxes) and the ranges (lower whiskers represent the regions of lower box - 1.5 * IQR (inter-quartile range), upper whisker represent the regions of upper box - 1.5 * IQR). Wilcoxon test were performed between AT+GCPs and ND+GCPs. ***p < 0.001.


C - F . Ccnd1 KD analysis . ( C , D ) Ccnd1 shRNAs together with H2B - EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 ( magenta ) and KI67 ( Cyan ) was performed the following day . Nuclei of electroporated cells were labeled with GFP ( yellow ) . Images of Control ( C ) and Ccnd1 KD ( D ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( E ) Proportions of ATOH1 + , KI67 + cells ( AT + GCPs ) among the electroporated cells were decreased in the Ccnd1 KD mice . ( F ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Student T - test was performed between Control ( N = 3 ) and Ccnd1 KD ( sh # 1 : N = 3 , sh # 2 : N = 3 ) mice . Bars and error bars are representing as Mean ± SEM . * * * p < 0 . 001 , N . S . : Not Significant . Number of cells : 52 , 64 and 107 cells from N = 3 mice ( scramble ) , 72 , 79 and 75 cells from N = 3 mice ( sh # 1 ) , 84 , 70 and 40 cells from N = 3 mice ( sh # 2 ) .

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

C-F. Ccnd1 KD analysis. (C, D) Ccnd1 shRNAs together with H2B-EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (Cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (C) and Ccnd1 KD (D). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (E) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were decreased in the Ccnd1 KD mice. (F) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=3) and Ccnd1 KD (sh#1: N=3, sh#2: N=3) mice. Bars and error bars are representing as Mean ± SEM. ***p < 0.001, N.S.: Not Significant. Number of cells: 52, 64 and 107 cells from N=3 mice (scramble), 72,79 and 75 cells from N=3 mice (sh#1), 84, 70 and 40 cells from N=3 mice (sh#2).


G - K . Over - expression analysis of Ccnd1 with or without Cdk4 . ( G , H , I ) Ccnd1 and Cdk4 expression plasmids were introduced to P5 mice EGL and immunostaining for ATOH1 ( magenta ) and KI67 ( cyan ) was performed the following day . Nuclei of electroporated cells were labeled with GFP ( yellow ) . Images of Control ( G ) , CCND1 over expression ( H ) and CCND1 and CDK4 over expression ( I ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( J ) Proportions of ATOH1 + , KI67 + cells ( AT + GCPs ) among the electroporated cells were not significantly changed between Control and CCND1 . Forced expression of CCND1 and CDK4 ( 4D ) significantly increased the proporations of AT + GCPs compared to Control . ( K ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Tukey - Kramer test was performed on Control ( N = 6 ) , CCND1 ( N = 4 ) , CCND1 and CDK4 ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * * p < 0 . 01 , N . S . : Not Significant . Number of cells : 52 , 64 , 107 , 244 , 127 and 146 from N = 6 mice ( Control ) , 126 , 137 , 80 and 336 from N = 4 mice ( Ccnd1 ) , 212 , 143 and 38 from N = 3 mice ( 4D ) .

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

G-K. Over-expression analysis of Ccnd1 with or without Cdk4. (G, H, I) Ccnd1 and Cdk4 expression plasmids were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (G), CCND1 over expression (H) and CCND1 and CDK4 over expression (I). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (J) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were not significantly changed between Control and CCND1. Forced expression of CCND1 and CDK4 (4D) significantly increased the proporations of AT+GCPs compared to Control. (K) Proportions of KI67+ cells (total GCPs) were not significantly changed. Tukey-Kramer test was performed on Control (N=6), CCND1 (N=4), CCND1 and CDK4 (N=3). Bars and error bars are representing as Mean ± SEM. **p < 0.01, N.S.: Not Significant. Number of cells: 52, 64, 107,244,127 and 146 from N=6 mice (Control), 126, 137, 80 and 336 from N=4 mice (Ccnd1), 212, 143 and 38 from N=3 mice (4D).


C . N2a cells transfected with Atoh1 - GST plasmids were treated with DMSO ( Control ) , Palbociclib ( Palb ) or Palb and 10 uM MG132 ( MG132 ) for 6hrs before harvest . Lysates of N2a were subjected to immunoblotting with anti - Atoh1 or β - actin . Quantification of relative ATOH1 to the β - ACTIN ( right panel ) . Tukey - Kramer test was performed between Control , Palb , MG132 . Bars and error bars are representing as Mean ± SEM from three trials of each condition . * * * p < 0 . 001 , * p < 0 . 05 , N . S . : Not Significant . Three slots of each experimental condition represent the three biological replicants of each condition .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. N2a cells transfected with Atoh1-GST plasmids were treated with DMSO(Control), Palbociclib (Palb) or Palb and 10 uM MG132 (MG132) for 6hrs before harvest. Lysates of N2a were subjected to immunoblotting with anti-Atoh1 or β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between Control, Palb, MG132. Bars and error bars are representing as Mean ± SEM from three trials of each condition. ***p < 0.001, *p < 0.05, N.S.: Not Significant. Three slots of each experimental condition represent the three biological replicants of each condition.


G . Atoh1 expression plasmids were transfected into N2a cells with Ccnd1 and Cdk4 expression plasmids ( 4D ) . N2a cells were treated with 10 nM , 100 nM , 1000 nM Palb for 6 hrs before harvest . Lysates from N2a cells were subjected to immunoblotting with anti - Atoh1 , p - S309 - Ab and β - actin . Quantification of relative ATOH1 to the β - ACTIN ( right panel ) . Tukey - Kramer test was performed between 0 , 10 , 100 , 1000nM . Bars and error bars are representing as Mean ± SEM from three trials of each condition . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant .

O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Atoh1 expression plasmids were transfected into N2a cells with Ccnd1 and Cdk4 expression plasmids (4D). N2a cells were treated with 10 nM, 100 nM, 1000 nM Palb for 6 hrs before harvest. Lysates from N2a cells were subjected to immunoblotting with anti-Atoh1, p-S309-Ab and β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between 0, 10, 100, 1000nM. Bars and error bars are representing as Mean ± SEM from three trials of each condition. **p < 0.01, *p < 0.05, N.S.: Not Significant.


B . Lysates of N2a cells transfected with either WT or a phospho - mimic form of ATOH1 ( S309D ) were immunoblotted with anti - Atoh1 or β - actin . Quantification of relative levels of ATOH1 to the β - ACTIN ( right panel ) . Mean ± s . e . m . Student T - test between WT and S309D was performed . * * * p < 0 . 001 . Three slots of each experimental condition represent the three biological replicants of each condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Lysates of N2a cells transfected with either WT or a phospho-mimic form of ATOH1 (S309D) were immunoblotted with anti-Atoh1 or β-actin. Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Mean± s.e.m. Student T-test between WT and S309D was performed. ***p < 0.001. Three slots of each experimental condition represent the three biological replicants of each condition.


Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1 . ( F - I ) Scramble ( Control ) , Atoh1 shRNA ( Atoh1 KD ) , shRNA and WT ( KD + WT ) or shRNA and S309A ( KD + S309A ) plasmids were introduced to P5 mice EGL with H2B - EGFP plasmid and immunostaining for NEUROD1 ( magenta ) was performed at P7 . Nuclei of electroporated cells were detected by GFP ( green ) . Arrows indicate the cells of interest which GFP signals are not co - localized with NEUROD1 ( AT + GCPs ) .

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

Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1. (F-I) Scramble (Control), Atoh1 shRNA (Atoh1 KD), shRNA and WT (KD + WT) or shRNA and S309A (KD + S309A) plasmids were introduced to P5 mice EGL with H2B-EGFP plasmid and immunostaining for NEUROD1(magenta) was performed at P7. Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest which GFP signals are not co-localized with NEUROD1 (AT+GCPs).


B - D . Overexpression analysis of PROX1 . ( B , C ) Prox1 expression plasmids were introduced to mouse P5 EGL and immunostaining for CCND1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected with GFP ( green ) . Images of Control ( B ) , PROX1 overexpression ( C ) . Arrow heads indicates the CCND1 positive cells . White lines are representing as pial surfaces . ( D ) The proportion of CCND1 - positive cells among the electroporated cells . Bars and error bars are representing as Mean ± SEM . Student T - test between Control ( N = 3 ) and PROX1 overexpression ( N = 3 ) . * * p < 0 . 01 . Number of cells 382 , 147 and 88 cells from N = 3 mice ( control ) , 187 , 108 and 94 cells from N = 3 mice ( Prox1 OE ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. Overexpression analysis of PROX1. (B, C) Prox1 expression plasmids were introduced to mouse P5 EGL and immunostaining for CCND1 (magenta) was performed the following day. Nuclei of electroporated cells were detected with GFP (green). Images of Control (B), PROX1 overexpression (C). Arrow heads indicates the CCND1 positive cells. White lines are representing as pial surfaces. (D) The proportion of CCND1-positive cells among the electroporated cells. Bars and error bars are representing as Mean ± SEM. Student T-test between Control (N=3) and PROX1 overexpression (N=3). **p < 0.01. Number of cells 382, 147 and 88 cells from N=3 mice (control), 187, 108 and 94 cells from N=3 mice (Prox1 OE).


E - G . Prox1 acute KO analysis . ( E , F ) Expression plasmids of Cre recombinase were introduced to P5 EGL of Prox1fl / fl mice and immunostaining for CCND1 ( magenta ) was performed at P8 . Nuclei of electroporated cells were labeled with GFP ( green ) . Images of H2B - GFP electroporation ( E , Control ) , Cre electroporation ( F , Cre ) . Arrows indicate the cells of interest where GFP signals are co - localized with CCND1 . ( G ) The proportions of CCND1 + cells among the electroporated cells . Bars and error bars are representing as Mean ± SEM . Student T - test between Control ( N = 3 ) and Cre ( N = 3 ) . * * p < 0 . 01 . Number of cells : 238 , 129 and 529 cells from N = 3 mice ( Control ) , 169 , 210 and 216 cells from N = 3 mice ( Cre ) .

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

E-G. Prox1 acute KO analysis. (E, F) Expression plasmids of Cre recombinase were introduced to P5 EGL of Prox1fl/fl mice and immunostaining for CCND1 (magenta) was performed at P8. Nuclei of electroporated cells were labeled with GFP (green). Images of H2B-GFP electroporation (E, Control), Cre electroporation (F, Cre). Arrows indicate the cells of interest where GFP signals are co-localized with CCND1. (G) The proportions of CCND1+ cells among the electroporated cells. Bars and error bars are representing as Mean ± SEM. Student T-test between Control (N=3) and Cre (N=3). **p < 0.01. Number of cells: 238, 129 and 529 cells from N=3 mice (Control), 169, 210 and 216 cells from N=3 mice (Cre).


H - K . Rescue experiment of Prox1 with Ccnd1 and Cdk4 - expressing plasmids . ( H - J ) Prox1 expressing plasmids were introduced with Ccnd1 and Cdk4 expression plasmids ( Prox1 + 4D ) to P5 EGL together with H2B - GFP plasmid . Immunostaining for ATOH1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected by GFP ( green ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( K ) Proportions of ATOH1 + cells ( AT + GCPs ) among the electroporated cells . The Tukey - Kramer test was performed on Control ( N = 6 ) , Prox1 ( N = 3 ) and Prox1 + 4D ( N = 4 ) mice . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant . Bars and error bars are representing as Mean ± SEM . Control value is the same as Fig 2G , K . Number of cells : 54 , 72 and 59 cells from N = 3 mice ( Prox1 ) , 26 , 53 , 68 and 53 cells from N = 4 mice ( Prox1 + 4D ) .

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

H-K. Rescue experiment of Prox1 with Ccnd1 and Cdk4-expressing plasmids. (H-J) Prox1 expressing plasmids were introduced with Ccnd1 and Cdk4 expression plasmids (Prox1+4D) to P5 EGL together with H2B-GFP plasmid. Immunostaining for ATOH1 (magenta) was performed the following day. Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (K) Proportions of ATOH1+ cells (AT+GCPs) among the electroporated cells. The Tukey-Kramer test was performed on Control (N=6), Prox1 (N=3) and Prox1+4D (N=4) mice. **p < 0.01, *p <0.05, N.S.: Not Significant. Bars and error bars are representing as Mean ± SEM. Control value is the same as Fig 2G,K. Number of cells: 54, 72 and 59 cells from N=3 mice (Prox1), 26, 53, 68 and 53 cells from N=4 mice (Prox1+4D).


L . ChIP assay in P7 mouse cerebellum with anti - Prox1 or goat IgG as a negative control . Primer set used in this experiment was designed to amplify Ccnd1 promoter region . M - R . Administration of Valproic acid ( VPA ) to electroporated mice . ( M ) Expression plasmids of Prox1 were introduced to P5 EGL and VPA or Saline was administered intraperitoneally 2 days after electroporation . Immunostaining for CCND1 ( magenta ) was performed the day following the last VPA treatment . Nuclei of electroporated cells were detected with GFP ( green ) . Arrows indicate the cells of interest where GFP signals are co - localized with CCND1 . ( N - Q ) Images of H2B - GFP with Saline ( N ) , PROX1 with Saline ( O ) , H2B - GFP with VPA ( P ) , and PROX1 with VPA ( Q ) . ( R ) The relative proportions of CCND1 + cells among the electroporated cells ( normalized to the Control ) . Tukey - Kramer test among Control + saline ( N = 4 ) , PROX1 + saline ( N = 3 ) , Control + VPA ( N = 3 ) and PROX1 + VPA ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant . Number of cells : 120 , 488 , 934 and 211 from N = 4 mice ( Control + saline ) , 630 , 356 and 545 from N = 3 mice ( Control + VPA ) , 363 , 127 and 219 from N = 3 mice ( Prox1 + saline ) , 110 , 116 and 202 from N = 3 mice ( Prox1 + VPA ) .

O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O 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-CONTROLLED_VAR O O

L. ChIP assay in P7 mouse cerebellum with anti-Prox1 or goat IgG as a negative control. Primer set used in this experiment was designed to amplify Ccnd1 promoter region. M-R. Administration of Valproic acid (VPA) to electroporated mice. (M) Expression plasmids of Prox1 were introduced to P5 EGL and VPA or Saline was administered intraperitoneally 2 days after electroporation. Immunostaining for CCND1 (magenta) was performed the day following the last VPA treatment. Nuclei of electroporated cells were detected with GFP (green). Arrows indicate the cells of interest where GFP signals are co-localized with CCND1. (N-Q) Images of H2B-GFP with Saline (N), PROX1 with Saline (O), H2B-GFP with VPA (P), and PROX1 with VPA (Q). (R) The relative proportions of CCND1+ cells among the electroporated cells (normalized to the Control).Tukey-Kramer test among Control+saline (N=4), PROX1+saline (N=3), Control +VPA (N=3) and PROX1 +VPA (N=3). Bars and error bars are representing as Mean ± SEM. **p < 0.01, *p <0.05, N.S.: Not Significant. Number of cells: 120, 488, 934 and 211 from N=4 mice (Control+saline), 630, 356 and 545 from N=3 mice (Control+VPA), 363, 127 and 219 from N=3 mice (Prox1+saline), 110, 116 and 202 from N=3 mice (Prox1+VPA).


H - J . Whole cerebellum lysates of P6 , P9 and P12 mice were subjected to immunoblotting with anti - pAtoh1 S309 , Atoh1 , Ccnd1 , Prox1 , β - actin . ( I ) The protein levels of CCND1 and PROX1 normalized by β - ACTIN . ( J ) Quantification of p -

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H-J. Whole cerebellum lysates of P6, P9 and P12 mice were subjected to immunoblotting with anti-pAtoh1 S309, Atoh1, Ccnd1, Prox1, β-actin. (I) The protein levels of CCND1 and PROX1 normalized by β-ACTIN. (J) Quantification of p-


L - P . Over - expression analysis of WT or S309D form of Atoh1 . ( L ) Expression plasmids of either WT or S309D form of Atoh1 were introduced to P10 mice EGL and immunostaining for KI67 ( magenta ) was performed in P16 mouse cerebellum . Nuclei of electroporated cells were detected by GFP ( green ) . Images of H2B - GFP ( M , Control ) , WT ( N ) and S309D ( O ) . Arrows indicate the cells of interest where GFP signals are co - localized with KI67 . ( GCPs ) . ( P ) Proportions of KI67 + cells among the electroporated cells . Tukey - Kramer test among Control ( N = 3 ) , Atoh1 WT ( N = 3 ) and Atoh1 S309D ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * p < 0 . 05 . Number of cells : 43 , 68 and 46 from N = 3 mice ( Control ) , 104 , 215 and 157 cells from N = 3 mice ( WT ) , 242 , 119 and 111 cells from N = 3 mice ( S309D ) .

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L-P. Over-expression analysis of WT or S309D form of Atoh1. (L) Expression plasmids of either WT or S309D form of Atoh1 were introduced to P10 mice EGL and immunostaining for KI67 (magenta) was performed in P16 mouse cerebellum. Nuclei of electroporated cells were detected by GFP (green). Images of H2B-GFP (M, Control), WT(N) and S309D (O). Arrows indicate the cells of interest where GFP signals are co-localized with KI67. (GCPs). (P) Proportions of KI67+ cells among the electroporated cells. Tukey-Kramer test among Control (N=3), Atoh1 WT (N=3) and Atoh1 S309D (N=3). Bars and error bars are representing as Mean ± SEM. *p <0.05. Number of cells: 43, 68 and 46 from N=3 mice (Control), 104, 215 and 157 cells from N=3 mice (WT), 242, 119 and 111 cells from N=3 mice (S309D).


D - F . Over - expression analysis of the activated form of β - catenin ( βcat S33Y ) . ( D , E ) Expression plasmids of βcat S33Y were introduced to P5 mice EGL and immunostaining for PROX1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected with GFP ( green ) . Images of H2B - GFP ( D , Control ) and βcat S33Y ( E , S33Y ) . Arrows indicate the cells of interest where GFP signals are co - localized with PROX1 . ( F ) Proportions of PROX1 - positive cells in electroporated cells . Bars and error bars are representing as Mean ± SEM . Student T - test between Control and S33Y was performed . * * * p < 0 . 001 . Number of cells : 89 , 90 and 55 cells from N = 3 mice ( Control ) , 32 , 111 and 56 cells from N = 3 mice ( S33Y ) .

D-F. Over-expression analysis of the activated form of β-catenin (βcat S33Y). (D, E) Expression plasmids of βcat S33Y were introduced to P5 mice EGL and immunostaining for PROX1 (magenta) was performed the following day. Nuclei of electroporated cells were detected with GFP (green). Images of H2B-GFP (D, Control) and βcat S33Y (E, S33Y). Arrows indicate the cells of interest where GFP signals are co-localized with PROX1. (F) Proportions of PROX1-positive cells in electroporated cells. Bars and error bars are representing as Mean± SEM. Student T-test between Control and S33Y was performed. ***p < 0.001. Number of cells: 89, 90 and 55 cells from N=3 mice (Control), 32, 111 and 56 cells from N=3 mice (S33Y).


J - L . Expression plasmids of βcat S33Y were introduced with or without Ccnd1 and Cdk4 expression plasmids ( 4D ) to P5 mice EGL and immunostaining for ATOH1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected by GFP ( green ) . Images of βcat S33Y ( J , S33Y ) and βcat S33Y with Ccnd1 and Cdk4 ( K ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . Proportions of ATOH1 + cells ( AT + GCPs ) among the electroporated cells . Control value is the same as Fig 2G - K . Bars and error bars are representing as Mean ± SEM . Tukey - Kramer test was performed between Control , S33Y and S33Y + 4D ( L ) . * * * p < 0 . 001 , N . S : Not Significant . Number of cells : 123 , 138 and 113 cells from N = 3 mice ( S33Y ) , 144 , 223 and 180 cells from N = 3 mice ( S33Y + 4D ) .

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J-L. Expression plasmids of βcat S33Y were introduced with or without Ccnd1 and Cdk4 expression plasmids (4D) to P5 mice EGL and immunostaining for ATOH1 (magenta) was performed the following day. Nuclei of electroporated cells were detected by GFP (green). Images of βcat S33Y (J, S33Y) and βcat S33Y with Ccnd1 and Cdk4 (K). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1.Proportions of ATOH1+ cells (AT+GCPs) among the electroporated cells. Control value is the same as Fig 2G-K. Bars and error bars are representing as Mean± SEM. Tukey-Kramer test was performed between Control, S33Y and S33Y+4D (L). ***p < 0.001, N.S: Not Significant. Number of cells: 123, 138 and 113 cells from N=3 mice (S33Y), 144, 223 and 180 cells from N=3 mice (S33Y+4D).


A . Assessment of protein levels in different AIFM1 knockout clones . Lysates from different AIFM1 knockout clones and wild type cells were analyzed by reducing SDS - PAGE and immunoblotting . MIA40 / CHCHD4 levels are not changed in AIFM1 knockout HEK293 cells .

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A. Assessment of protein levels in different AIFM1 knockout clones. Lysates from different AIFM1 knockout clones and wild type cells were analyzed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 levels are not changed in AIFM1 knockout HEK293 cells.


D . Assessment of protein levels in HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1 - HA ( expression of AIFM1 - HA induced for one day ) . Different amounts of lysates from the indicated cells were analyzed by reducing SDS - PAGE and immunoblotting . While levels of MIA40 were largely unchanged , levels of many MIA40 / CHCHD4 substrates were diminished in AIFM1 knockout cells . N = 3 biological replicates .

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D. Assessment of protein levels in HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA (expression of AIFM1-HA induced for one day). Different amounts of lysates from the indicated cells were analyzed by reducing SDS-PAGE and immunoblotting. While levels of MIA40 were largely unchanged, levels of many MIA40/CHCHD4 substrates were diminished in AIFM1 knockout cells. N = 3 biological replicates.


F . BN - PAGE analyses of HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1 - HA or AIFM1 . Complex I was resolved from isolated mitochondria with blue native electrophoresis ( BN - PAGE ) followed by immunoblotting or in gel determination of the complex I specific activity . Levels of complex I were decreased in AIFM1 knockout cells . N = 3 biological

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F. BN-PAGE analyses of HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA or AIFM1. Complex I was resolved from isolated mitochondria with blue native electrophoresis (BN-PAGE) followed by immunoblotting or in gel determination of the complex I specific activity. Levels of complex I were decreased in AIFM1 knockout cells. N = 3 biological replicates


A . In organello import assay with NDUFS5 , NDUFB7 and as control the matrix protein SOD2 . In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 and AIFM1 knockout cells . Non - imported proteins were removed by treatment with Proteinase K . Imported proteins were analyzed by reducing SDS - PAGE and autoradiography . Signals were quantified using ImageQuantTL and the amount of imported protein was plotted . NDUFS5 import is strongly affected by loss of AIFM1 . Import of NDUFB7 is less affected and SOD2 import remained unaffected . This is despite the fact that the core domains important for oxidative folding of NDUFS5 an NDUFB7 are very similar .

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A. In organello import assay with NDUFS5, NDUFB7 and as control the matrix protein SOD2. In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 and AIFM1 knockout cells. Non-imported proteins were removed by treatment with Proteinase K. Imported proteins were analyzed by reducing SDS-PAGE and autoradiography. Signals were quantified using ImageQuantTL and the amount of imported protein was plotted. NDUFS5 import is strongly affected by loss of AIFM1. Import of NDUFB7 is less affected and SOD2 import remained unaffected. This is despite the fact that the core domains important for oxidative folding of NDUFS5 an NDUFB7 are very similar.


B . Oxidation kinetics of endogenous NDUFB7 and NDUFS5 . HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1 - HA were analyzed by pulse - chase experiments coupled to redox state determination . To this end , cells were incubated with 35S - Met for 5 min ( pulse ) . After removal of 35S - Met , cells were left in " cold " Met for different chase times . Further oxidation was stopped by addition of trichloroacetic acid ( TCA ) . Free thiols but not thiols in disulfide bonds were modified using mmPEG24 and then MIA40 / CHCHD4 substrates were enriched by immunoprecipitation ( IP ) , and analyzed by non - reducing SDS - PAGE and autoradiography . Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells . NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed . N = 4 biological replicates for NDUFB7 and NDUFS5 .

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B. Oxidation kinetics of endogenous NDUFB7 and NDUFS5. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analyzed by pulse-chase experiments coupled to redox state determination. To this end, cells were incubated with 35S-Met for 5 min (pulse). After removal of 35S-Met, cells were left in "cold" Met for different chase times. Further oxidation was stopped by addition of trichloroacetic acid (TCA). Free thiols but not thiols in disulfide bonds were modified using mmPEG24 and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells. NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed. N = 4 biological replicates for NDUFB7 and NDUFS5.


C . Synthesis of NDUFS5 and NDUFB7 in wild type and AIFM1 knockout cells . Cells were incubated with 35S - Met for 5 min ( pulse ) , and then MIA40 / CHCHD4 substrates were enriched by immunoprecipitation ( IP ) , and analyzed by non - reducing SDS - PAGE and autoradiography . NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells .

C. Synthesis of NDUFS5 and NDUFB7 in wild type and AIFM1 knockout cells. Cells were incubated with 35S-Met for 5 min (pulse), and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells.


D . Interaction between endogenous MIA40 / CHCHD4 and its substrates in wild type and AIFM1 knockout cells . Cells were subjected to native immunoprecipitation ( IP ) against endogenous MIA40 / CHCHD4 . Precipitates were analyzed by SDS - PAGE and immunoblotting against the indicated proteins . In AIFM1 knockout cell interaction between MIA40 / CHCHD4 and its substrates is strongly affected with the interaction between MIA40 / CHCHD4 and NDUFS5 almost completely abolished . This is despite the fact that NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells , indicating that lowered steady state levels of these proteins are consequence of a lacking interaction with MIA40 / CHCHD4 rather than the cause . Quantification using Image lab . Data from 2 experiments were combined and standard deviations are presented . Asterisk indicates background band orange arrow indicates signal of NDUFS5 and NDUFB7 in AIFM1 KO cells , respectively .

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D. Interaction between endogenous MIA40/CHCHD4 and its substrates in wild type and AIFM1 knockout cells. Cells were subjected to native immunoprecipitation (IP) against endogenous MIA40/CHCHD4. Precipitates were analyzed by SDS-PAGE and immunoblotting against the indicated proteins. In AIFM1 knockout cell interaction between MIA40/CHCHD4 and its substrates is strongly affected with the interaction between MIA40/CHCHD4 and NDUFS5 almost completely abolished. This is despite the fact that NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells, indicating that lowered steady state levels of these proteins are consequence of a lacking interaction with MIA40/CHCHD4 rather than the cause. Quantification using Image lab. Data from 2 experiments were combined and standard deviations are presented. Asterisk indicates background band orange arrow indicates signal of NDUFS5 and NDUFB7 in AIFM1 KO cells, respectively.


A . Assessment of MIA40 / CHCHD4 variant - AIFM1 interaction . The indicated MIA40 / CHCHD4 - Strep variants were affinity precipitated ( AP ) under native conditions after stopping thiol - disulfide exchange reactions by NEM incubation . Precipitates were tested for AIFM1 , ALR and MIA40 / CHCHD4 by reducing SDS - PAGE and immunoblotting . 1 % of the total lysate was loaded as input control . M , mock control not expressing MIA40 - Strep . Both , wildtype MIA40 / CHCHD4 and MTS - MIA40 / CHCHD4 but not MIA40 / CHCHD4 ∆ 1 - 40 variants coprecipitated AIFM1 . All mitochondria - localized variants precipitate ALR indicating redox functionality . Arrowhead indicates endogenous MIA40 / CHCHD4 .

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A. Assessment of MIA40/CHCHD4 variant-AIFM1 interaction. The indicated MIA40/CHCHD4-Strep variants were affinity precipitated (AP) under native conditions after stopping thiol-disulfide exchange reactions by NEM incubation. Precipitates were tested for AIFM1, ALR and MIA40/CHCHD4 by reducing SDS-PAGE and immunoblotting. 1% of the total lysate was loaded as input control. M, mock control not expressing MIA40-Strep. Both, wildtype MIA40/CHCHD4 and MTS-MIA40/CHCHD4 but not MIA40/CHCHD4∆1-40 variants coprecipitated AIFM1. All mitochondria-localized variants precipitate ALR indicating redox functionality. Arrowhead indicates endogenous MIA40/CHCHD4.


Emetine - chase experiments to assess the stability of the AIFM1 / CHCHD4 complex . HEK293 cells stably expressing MIA40 / CHCHD4 - Strep were treated for the indicated times with the ribosome inhibitor emetine . Then , cells were lysed under native conditions ( triton X - 100 ) , and MIA40 / CHCHD4 - Strep ( B ) were precipitated . Precipitates were analysed by reducing SDS - PAGE and immunoblotting . MIA40 / CHCHD4 interact over a period of at least 6 - 8 hours while the two MIA40 / CHCHD4 substrates , NDUFB7 and AK2 , are quickly released from MIA40 / CHCHD4 ( B , gray box ) . AP , affinity precipitation ; asterisk , background band .

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Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. HEK293 cells stably expressing MIA40/CHCHD4-Strep were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and MIA40/CHCHD4-Strep (B) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 interact over a period of at least 6-8 hours while the two MIA40/CHCHD4 substrates, NDUFB7 and AK2, are quickly released from MIA40/CHCHD4 (B, gray box). AP, affinity precipitation; asterisk, background band.


C . Emetine - chase experiments to assess the stability of the AIFM1 / CHCHD4 complex . AIFM1 knockout cells stably expressing AIFM1 - HA ( C ) were treated for the indicated times with the ribosome inhibitor emetine . Then , cells were lysed under native conditions ( triton X - 100 ) , and AIFM1 - HA ( C ) were precipitated . Precipitates were analysed by reducing SDS - PAGE and immunoblotting . MIA40 / CHCHD4 and AIFM1 interact over a period of at least 6 - 8 hours AP , affinity precipitation ; asterisk , background band .

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C. Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. AIFM1 knockout cells stably expressing AIFM1-HA (C) were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and AIFM1-HA (C) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 and AIFM1 interact over a period of at least 6-8 hours AP, affinity precipitation; asterisk, background band.


Eluted fractions were subjected to SDS - PAGE and TCE staining ( E ) . AIFM1 and full - length MIA40 / CHCHD4 migrate together indicating formation of a complex . MIA40 / CHCHD4 was present in excess over AIFM1 resulting in the presence of free MIA40 / CHCHD4 . AIFM1 and ∆ 1 - 40 MIA40 / CHCHD4 do not migrate together emphasizing the importance of the N - terminal amino acids in MIA40 for interaction wth AIFM1 .

Eluted fractions were subjected to SDS-PAGE and TCE staining (E). AIFM1 and full-length MIA40/CHCHD4 migrate together indicating formation of a complex. MIA40/CHCHD4 was present in excess over AIFM1 resulting in the presence of free MIA40/CHCHD4. AIFM1 and ∆1-40 MIA40/CHCHD4 do not migrate together emphasizing the importance of the N-terminal amino acids in MIA40 for interaction wth AIFM1.