( I , J ) Statistical of Ki67 ( I ) CC3 ( J ) activity in ADC IMA lung sections of vehicle phenformin ( Phen ) groups . numbers of tumors analyzed over vehicle group phenformin groups for ADC IMA were 17 , 8 , 9 for Ki67 , 10 , 10 , 7 for CC3 . Results were shown as mean ± . Significance was calculated by one - way ANOVA with Dunnett ' s multiple comparisons . * * p = 0 . 0010 ( I ) , * * * p = 0 . 0009 ( I ) , * p = 0 . 0427 ( J ) .

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(I, J) Statistical analysis of Ki67 (I) and CC3 (J) activity in ADC and IMA lung sections of vehicle and phenformin (Phen) groups. The numbers of tumors analyzed over vehicle group and phenformin groups for ADC and IMA were 17, 8, 9 for Ki67, 10, 10, 7 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0010 (I), ***p=0.0009 (I), *p=0.0427(J).


( E , F ) Statistical of Ki67 ( E ) CC3 ( F ) reactivity in lung sections of KLN mice following vehicle , LY - 411575 treatment or a combinational therapy of phenformin LY - 411575 . numbers of tumors analyzed over vehicle , phenformin combinational therapy groups were 20 , 30 , 13 for Ki67 , 14 , 11 , 9 for CC3 . Results were shown as mean ± . Significance was calculated by one - way ANOVA with Dunnett ' s multiple comparisons . * * p = 0 . 0014 ( E ) , * * * p = 0 . 0001 ( F ) .

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(E, F) Statistical analysis of Ki67 (E) and CC3 (F) reactivity in lung sections of KLN mice following vehicle, LY-411575 treatment or a combinational therapy of phenformin and LY-411575. The numbers of tumors analyzed over vehicle, phenformin and combinational therapy groups were 20, 30, 13 for Ki67, 14, 11, 9 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0014 (E), ***p=0.0001 (F).


B - Western blot verifying expression of CTD variants RPB1 - 52R RPB1 - 25R in U2OS in whole extracts . U1 snRNP was used as a loading control .

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B- Western blot verifying the expression of the CTD variants RPB1-52R and RPB1-25R in U2OS cells in whole cell extracts. U1 snRNP was used as a loading control.


C - Changes in synthesis upon CTD shortening . MA plot showing synthesis changes in TT - seq datasets upon CTD shortening in U2OS in steady state conditions . RPB1 - 52R are used as a control data was normalized using spike - in . 16214 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red . 473 genes were significantly upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) 704 genes were significantly downregulated ( adjusted p value < 0 . 05 , log2 fold change ≤ 1 ) .

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C- Changes in RNA synthesis upon CTD shortening. MA plot showing RNA synthesis changes in TT-seq datasets upon CTD shortening in U2OS cells in steady state conditions. RPB1-52R cells are used as a control and the data was normalized using spike-in counts. 16214 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red. 473 genes were significantly upregulated (adjusted p value < 0.05, log2 fold change >=1) and 704 genes were significantly downregulated (adjusted p value < 0.05, log2 fold change ≤1).


D - Box plot of estimated synthesis rates of expressed RefSeq transcripts ( 12014 transcripts ) on TT - seq - seq datasets in RPB1 - 52R RPB1 - 25R . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range .

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D- Box plot of estimated RNA synthesis rates of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


E - Box plot of estimated degradation rates of expressed RefSeq transcripts ( 12014 transcripts ) on TT - seq - seq datasets in RPB1 - 52R RPB1 - 25R . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range .

E- Box plot of estimated RNA degradation rates of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


F - Box plot of estimated half - lives of expressed RefSeq transcripts ( 12014 transcripts ) on TT - seq - seq datasets in RPB1 - 52R RPB1 - 25R . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range .

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F- Box plot of estimated RNA half-lives of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


A - Box plots showing ratios of spliced TT - seq reads over total unspliced spliced TT - seq reads for constitutive 5 ' SSs 3 ' SSs detected in major isoforms in RPB1 - 52R RPB1 - 25R . p value was calculated using Mann - Whitney U . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed . B - D - Box plots showing ratios of spliced TT - seq reads over total unspliced spliced TT - seq reads in RPB1 - 52R RPB1 - 25R for constitutive 5 ' SSs 3 ' SSs detected in major isoforms on intron position in transcript : ( B ) first intron junction , ( C ) intermediate intron junctions ( D ) last intron junctions . p values were calculated using Mann - Whitney U . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range .

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A- Box plots showing ratios of spliced TT-seq reads over total unspliced and spliced TT-seq reads for all constitutive 5'SSs and 3'SSs detected in major RNA isoforms in RPB1-52R and RPB1-25R cells. p value was calculated using Mann-Whitney U test. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. B-D- Box plots showing ratios of spliced TT-seq reads over total unspliced and spliced TT-seq reads in RPB1-52R and RPB1-25R cells for all constitutive 5'SSs and 3'SSs detected in major RNA isoforms based on intron position in the transcript: (B) first intron junction, (C) intermediate intron junctions and (D) last intron junctions. p values were calculated using Mann-Whitney U test. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


E - Differential mRNA isoform usage upon CTD shortening . Scatter plot showing read for mRNA isoforms detected in fraction of RPB1 - 52R RPB1 - 25R using long read sequencing . 47416 isoforms were detected , RPB1 - 52R condition was used as a control . 335 isoforms show differential expression ( p value < 0 . 05 , Fischer ' s exact ) .

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E- Differential mRNA isoform usage upon CTD shortening. Scatter plot showing read counts for mRNA isoforms detected in the chromatin fraction of RPB1-52R and RPB1-25R cells using long read sequencing. 47416 isoforms were detected, RPB1-52R condition was used as a control. 335 isoforms show differential expression (p value < 0.05, Fischer's exact test).


A - CTD shortening leads to a decreased Pol II occupancy in promoter - proximal region . Median coverage plot of PRO - seq signal at genes encoding major isoforms . Solid lines represent median signal shaded refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

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A- CTD shortening leads to a decreased Pol II occupancy in promoter-proximal region. Median coverage plot of PRO-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


B , C - Pol II pause position remains unchanged upon CTD shortening . Histogram of estimated positions of paused Pol II in RPB1 - 52R RPB1 - 25R at genes encoding major isoforms with constitutive first exon longer than 100 bp .

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B,C- Pol II pause position remains unchanged upon CTD shortening. Histogram of estimated positions of paused Pol II in RPB1-52R and RPB1-25R cells at genes encoding major isoforms with constitutive first exon longer than 100 bp.


D - Median coverage plot of TT - seq signal at genes encoding major isoforms in RPB1 - 52R RPB1 - 25R . Solid lines represent median signal shaded refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

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D- Median coverage plot of TT-seq signal at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


E - Box plot showing productive transcription initiation rate in RPB1 - 52R RPB1 - 25R for genes encoding major isoforms . p value = 0 . 13 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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E- Box plot showing productive transcription initiation rate in RPB1-52R and RPB1-25R cells for genes encoding major isoforms. p value=0.13 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


F - Box plot showing Pol II pausing duration in RPB1 - 52R RPB1 - 25R for genes encoding major isoforms with constitutive first exon longer than 100 bp . p value < 2 . 2e - 16 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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F- Box plot showing Pol II pausing duration in RPB1-52R and RPB1-25R cells for genes encoding major isoforms with constitutive first exon longer than 100 bp. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


G - Median scaled coverage plot of TT - seq signal at genes encoding major isoforms . Solid lines represent median signal shaded refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

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G- Median scaled coverage plot of TT-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


H - Pol II velocity remains unchanged upon CTD shortening . Metagene profile of transcription elongation velocity at 4480 genes longer than 10 kb encoding major isoforms . Solid lines represent median signal shaded refer to 95 % bootstrap confidence intervals .

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H- Pol II velocity remains unchanged upon CTD shortening. Metagene profile of transcription elongation velocity at 4480 genes longer than 10 kb encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals.


I - Median coverage plot of TT - seq signal centered at PAS at genes encoding major isoforms in RPB1 - 52R RPB1 - 25R . Solid lines represent median signal shaded refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

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I- Median coverage plot of TT-seq signal centered at PAS at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


J - Box plot showing of poly ( A ) tail in RPB1 - 52R RPB1 - 25R measured using Nanopore sequening data . p value < 2 . 2e - 16 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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J - Box plot showing length of the poly(A) tail in RPB1-52R and RPB1-25R cells measured using Nanopore sequening data. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


K - Box plot showing from polyadenylation site ( PAS ) to transcription termination site ( TTS ) in RPB1 - 52R RPB1 - 25R for 1958 genes encoding major isoforms . p value = 0 . 8 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range .

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K- Box plot showing distance from the polyadenylation site (PAS) to the transcription termination site (TTS) in RPB1-52R and RPB1-25R cells for 1958 genes encoding major isoforms. p value= 0.8 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


B - Box plots showing synthesis ( RPKs ) of putative eRNAs annotated using TT - seq data in RPB1 - 52R RPB1 - 25R . p value = 9 . 27e - 103 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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B- Box plots showing RNA synthesis levels (RPKs) of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. p value =9.27e-103 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


C - Box plots showing lengths of putative eRNAs annotated using TT - seq data in RPB1 - 52R RPB1 - 25R . p value = 0 . 000519 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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C- Box plots showing lengths of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. p value =0.000519 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


D - Histogram showing a distribution of number of putative eRNAs paired to genes in RPB1 - 52R RPB1 - 25R .

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D - Histogram showing a distribution of number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells.


A - Changes in synthesis in response to 15 min of TPA treatment . MA plot showing synthesis changes in TT - seq datasets upon 15 min treatment with TPA ( 200 nM ) in RPB1 - 52R RPB1 - 25R . DMSO treatment of respective was used as a control data was normalized using library normalization . 14636 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red ( adjusted p value < 0 . 05 , log2 fold change > = 1 for upregulated genes adjusted p value < 0 . 05 , log2 fold change ≤ 1 for downregulated genes ) . B - Box plots showing log2 fold change of genes significantly upregulated upon 15 min treatment with 200 nM TPA in RPB1 - 52R RPB1 - 25R ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) . P value = 0 . 02257 ( Mann - Whitney ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed . C - Changes in synthesis in response to 30 min of TPA treatment . MA plot showing synthesis changes in TT - seq datasets upon 30 min treatment with TPA ( 200 nM ) in RPB1 - 52R RPB1 - 25R . DMSO treatment of respective was used as a control data was normalized using library normalization . 14636 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red ( adjusted p value < 0 . 05 , log2 fold change > = 1 for upregulated genes adjusted p value < 0 . 05 , log2 fold change ≤ 1 for downregulated genes ) D - Box plots showing log2 fold change in synthesis of genes significantly upregulated upon 30 min treatment with 200 nM TPA in RPB1 - 52R RPB1 - 25R ( adjusted p value < 0 . 05 , log2 fold chage > = 1 ) . P value = 0 . 001749 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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 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 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 B-CONTROLLED_VAR 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 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

A- Changes in RNA synthesis in response to 15 min of TPA treatment. MA plot showing RNA synthesis changes in TT-seq datasets upon 15 min treatment with TPA (200 nM) in RPB1-52R and RPB1-25R cells. DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization. 14636 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red (adjusted p value < 0.05, log2 fold change >=1 for upregulated genes and adjusted p value < 0.05, log2 fold change ≤1 for downregulated genes ). B- Box plots showing log2 fold change of genes significantly upregulated upon 15 min treatment with 200 nM TPA in RPB1-52R and RPB1-25R cells (adjusted p value < 0.05, log2 fold change >=1). P value =0.02257 (Mann-Whitney test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. C- Changes in RNA synthesis in response to 30 min of TPA treatment. MA plot showing RNA synthesis changes in TT-seq datasets upon 30 min treatment with TPA (200 nM) in RPB1-52R and RPB1-25R cells. DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization. 14636 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red (adjusted p value < 0.05, log2 fold change >=1 for upregulated genes and adjusted p value < 0.05, log2 fold change ≤1 for downregulated genes) D- Box plots showing log2 fold change in RNA synthesis of genes significantly upregulated upon 30 min treatment with 200 nM TPA in RPB1-52R and RPB1-25R cells (adjusted p value < 0.05, log2 fold chage >=1). P value =0.001749 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


A - Histogram showing distribution of a number of putative eRNAs paired to genes in RPB1 - 52R RPB1 - 25R upon 15 30 min TPA treatment ( 200 nM ) as well as in DMSO controls .

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A- Histogram showing the distribution of a number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells upon 15 and 30 min TPA treatment (200 nM) as well as in the DMSO controls.


B - Box plots showing synthesis ( RPK ) of putative eRNAs paired with genes upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) in RPB1 - 52R RPB1 - 25R upon 15 min of TPA treatment ( 200 nM ) . p value = 0 . 000732 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed . C - Box plots showing synthesis ( RPK ) of putative enhancer eRNAs paired with genes upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) in RPB1 - 52R RPB1 - 25R upon 30 min of TPA treatment ( 200 nM ) . p value = 2 . 77e - 5 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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B- Box plots showing RNA synthesis levels (RPK) of putative eRNAs paired with genes upregulated (adjusted p value < 0.05, log2 fold change >=1) in RPB1-52R and RPB1-25R cells upon 15 min of TPA treatment (200 nM). p value = 0.000732 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. C- Box plots showing RNA synthesis levels (RPK) of putative enhancer eRNAs paired with genes upregulated (adjusted p value < 0.05, log2 fold change >=1) in RPB1-52R and RPB1-25R cells upon 30 min of TPA treatment (200 nM). p value = 2.77e-5 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


( A ) WT or Mfn2 KO MEFs ( Mfn2 KO ) were treated with 1 μM Tg for 12 h then processed for EM visualization of ER . Scale bar : 1 μm .

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(A) WT or Mfn2 KO MEFs (Mfn2 KO cells) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology. Scale bar: 1 μm.


( B ) EM of Tg ‐ treated Mfn2 KO show accumulation of ER stacking . Scale bar : 1 μm .

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(B) EM images of Tg‐treated Mfn2 KO cells show accumulation of ER membrane stacking. Scale bar: 1 μm.


( C ) WT , Mfn2 KO or Mfn1 KO were transfected with Sec61β ‐ GFP plasmid treated with 1 μM Tg for 24 h . Confocal microscopy show ER vacuolization in Mfn2 KO treated with Tg . Scale bar : 10 μm . Insets show × 10 zoomed . Scale bar : 5 μm .

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(C) WT, Mfn2 KO or Mfn1 KO cells were transfected with the Sec61β‐GFP plasmid and treated with 1 μM Tg for 24 h. Confocal microscopy images show ER vacuolization in Mfn2 KO cells treated with Tg. Scale bar: 10 μm. Insets show × 10 zoomed images. Scale bar: 5 μm.


( D ) WT Mfn2 KO were treated with Tg 1 μM for 3 h then incubated with brefeldin A ‐ bodipy to stain ER Golgi . Representative flow ‐ cytometry histograms ( upper panel ) . Mean was used to quantify ER expansion ( n = 5 ) ( lower panel ) . Data are mean ± s . e . m . * P0 . 05 versus WT group .

O O O O B-CONTROLLED_VAR 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

(D) WT and Mfn2 KO cells were treated with Tg 1 μM for 3 h and then incubated with brefeldin A‐bodipy to stain ER and Golgi. Representative flow‐cytometry histograms (upper panel). Mean fluorescence intensity was used to quantify ER expansion (n=5) (lower panel). Data are mean±s.e.m. *P0.05 versus WT group.


( A ) WT Mfn2 KO were treated with 1 μM Tg for 12 or 24 h . Total caspase 3 were detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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 B-MEASURED_VAR I-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

(A) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h. Total and cleaved caspase 3 levels were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( B , C ) WT Mfn2 KO were treated with 0 . 5 μg / ml tunicamycin ( Tm ) , 100 ng / ml brefeldin A ( Bref ) , or 1 μM Tg for 24 h . Total caspase 3 were detected by western blot ( B ) caspase activity ( C ) by measurement of DEVD ‐ AFC substrate processing . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(B, C) WT and Mfn2 KO cells were treated with 0.5 μg/ml tunicamycin (Tm), 100 ng/ml brefeldin A (Bref), or 1 μM Tg for 24 h. Total and cleaved caspase 3 levels were detected by western blot (B) and caspase activity (C) by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( D ) Flow ‐ cytometry of Sub G1 fragmentation in methanol ‐ fixed WT Mfn2 KO after incubation with or without 1 μM Tg for 24 h . Data are given as mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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 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

(D) Flow‐cytometry analysis of the Sub G1 DNA fragmentation in methanol‐fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group.


( E ) Scr ( stably expressing scrambled shRNA ) Mfn2 knockdown ( KD ) ( stably expressing shRNA directed against Mfn2 ) 3T3 ‐ L1 fibroblasts were incubated in presence or absence of 1 μM Tg for 24 h . Total caspase 3 were detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(E) Scr (stably expressing scrambled shRNA) and Mfn2 knockdown (KD) (stably expressing shRNA directed against Mfn2) 3T3‐L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h. Total and cleaved caspase 3 were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( F ) WT Mfn2 KO were treated with 1 μM Tg for 24 h . Lactate dehydrogenase ( LDH ) release was analyzed by flow cytometry to assess necrotic . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(F) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h. Lactate dehydrogenase (LDH) release was analyzed by flow cytometry to assess necrotic cell death. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( G ) WT Mfn2 KO were incubated for 24 h with or without 1 μM Tg in presence or absence of z ‐ VAD ‐ fmk stained for annexin V / PI . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT + Tg group .

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

(G) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z‐VAD‐fmk and stained for annexin V/PI. Data are mean±s.e.m. (n=4). *P0.05 versus WT+Tg group.


( H ) Mfn2 KO transfected with pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h ( 3 h of pre ‐ incubation with CHX ) . Florescence microscopy show that CHX prevents vacuolization . Scale bar : 10 μm .

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

(H) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h (3 h of pre‐incubation with CHX). Florescence microscopy images show that CHX prevents cytoplasmic vacuolization. Scale bar: 10 μm.


( I ) WT ( black circles ) Mfn2 KO ( circles ) were incubated with 1 μM Tg for varying times , ALIX was detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . Source data for this figure is available on online supplementary information page .

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

(I) WT (black circles) and Mfn2 KO cells (white circles) were incubated with 1 μM Tg for varying times, and ALIX protein was detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


( A , upper panel ) WT ( black circles ) or Mfn2 KO ( circles ) were treated with 1 μM Tg for a range of times . ( A , lower ) Densitometric . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group ; # P0 . 05 versus WT non ‐ Tg ‐ treated group .

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 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 O O O

(A, upper panel) WT (black circles) or Mfn2 KO cells (white circles) were treated with 1 μM Tg for a range of times. (A, lower) Densitometric quantification. Data are mean±s.e.m. (n=3). *P0.05 versus WT group; #P0.05 versus WT non‐Tg‐treated group.


( B , upper panel ) WT or Mfn2 KO were treated with Tg for 0 , 6 or 12 h , in presence or absence of Bafilomycin ( Baf , 100 nM ) . LC3b ‐ I LC3b ‐ II expression was measured by western blot . ( B , lower panel ) Densitometric of LC3b ‐ II ( relative to tubulin ) . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT + Baf + Tg group .

O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O B-MEASURED_VAR 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 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, upper panel) WT or Mfn2 KO cells were treated with Tg for 0, 6 or 12 h, in the presence or absence of Bafilomycin (Baf, 100 nM). LC3b‐I and LC3b‐II expression was measured by western blot. (B, lower panel) Densitometric quantification of LC3b‐II levels (relative to tubulin). Data are mean±s.e.m. (n=3). *P0.05 versus WT+Baf+Tg group.


( C ) WT or Mfn2 KO stably expressing mCherry ‐ GFP ‐ LC3b were treated with 1 μM Tg , 100 ng / ml Brefeldin ( Bref ) , or 0 . 5 μg / ml tunicamycin ( Tm ) for 24 h examined by confocal microscopy . Scale bar : 10 μm .

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

(C) WT or Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b were treated with 1 μM Tg, 100 ng/ml Brefeldin (Bref), or 0.5 μg/ml tunicamycin (Tm) for 24 h and examined by confocal microscopy. Scale bar: 10 μm.


( D ) of mCherry + / GFP ‐ ( mCherry ‐ LC3 ) red per ( a measure of acidic autophagosomes ) is shown . Data are mean ± s . e . m . ( n = 3 ; 100 were analyzed per experiment group ) . * P0 . 05 versus WT group .

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

(D) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus WT group.


( E ) Acidic compartments of WT or Mfn2 KO were stained with Lysotracker Green analyzed by flow cytometry . Data are given as mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(E) Acidic compartments of WT or Mfn2 KO cells were stained with Lysotracker Green and analyzed by flow cytometry. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group.


( F ) LAMP1 expression was immunodetected in WT Mfn2 KO in basal conditions .

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

(F) LAMP1 expression was immunodetected in WT and Mfn2 KO cells in basal conditions.


( G ) Expression of Beclin ‐ 1 or LC3b transcripts in WT or Mfn2 KO treated with or without 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . Source data for this figure is available on online supplementary information page .

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

(G) Expression of Beclin‐1 or LC3b transcripts in WT or Mfn2 KO cells treated with or without 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


( B ) Immunodetection of p ‐ PERK , PERK , GADD34 , p ‐ eIF2α , eIF2α , ATF4 , CHOP , XBP ‐ 1s in WT Mfn2 KO treated with 1 μM Tg for times indicated .

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

(B) Immunodetection of p‐PERK,PERK, GADD34, p‐eIF2α, eIF2α, ATF4, CHOP, and XBP‐1s in WT and Mfn2 KO cells treated with 1 μM Tg for the times indicated.


( C ) Densitometric quantifications . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

O O O O O O 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) Densitometric quantifications. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( D ) Immunodetection of p ‐ eIF2α , eIF2α , ATF4 in WT Mfn2 KO treated with 0 . 5 μg / ml Tunicamycin ( Tm ) for times indicated .

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

(D) Immunodetection of p‐eIF2α, eIF2α, and ATF4 in WT and Mfn2 KO cells treated with 0.5 μg/ml Tunicamycin (Tm) for the times indicated.


( E ) Transcriptional activity driven by ATF6 . WT Mfn2 KO were co ‐ transfected with 5xATF6 ‐ GL3 TK ‐ Renilla plasmids treated with 1 μM Tg for 24 h after transfection . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT group . Source data for this figure is available on online supplementary information page .

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

(E) Transcriptional activity driven by ATF6. WT and Mfn2 KO cells were co‐transfected with the 5xATF6‐GL3 and TK‐Renilla plasmids and treated with 1 μM Tg for 24 h after transfection. Data are mean±s.e.m. (n=4). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


( A ) Immunodetection of PERK in WT Mfn2 KO stably expressing a scrambled shRNA ( Scr ) or a shRNA directed against PERK ( PERK KD ) .

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

(A) Immunodetection of PERK in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed against PERK (PERK KD).


( B ) Caspase activity was detected by measurement of DEVD - AFC substrate processing in WT or Mfn2 KO subjected to PERK silencing treated with 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group .

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

(B) Caspase activity was detected by measurement of DEVD-AFC substrate processing in WT or Mfn2 KO cells subjected to PERK silencing and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.


( C , D ) Total caspase 3 were detected by western blot . Densitometric is shown in D . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group .

O O O O O O B-MEASURED_VAR I-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

(C, D) Total and cleaved caspase 3 levels were detected by western blot. Densitometric quantification is shown in D. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.


( E , F ) Immunodetection of LC3b ‐ I ‐ II in WT Mfn2 KO subjected to PERK silencing treated as indicated with Tg or Baf ( 1 μM Tg ; 100 nM Baf ) for 6 h . Densitometric is shown in F . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group . Source data for this figure is available on online supplementary information page .

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

(E, F) Immunodetection of LC3b‐I and ‐II in WT and Mfn2 KO cells subjected to PERK silencing and treated as indicated with Tg or Baf (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.


( A ) Immunodetection of XBP ‐ 1s in WT Mfn2 KO stably expressing a scrambled shRNA ( Scr ) or a shRNA directed to XBP ‐ 1 ( XBP ‐ 1 KD ) , treated with Tg for 6 h .

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

(A) Immunodetection of XBP‐1s in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed to XBP‐1 (XBP‐1 KD), and treated with Tg for 6 h.


( B ) Caspase activity was detected by measurement of DEVD ‐ AFC substrate processing . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . # P0 . 05 versus Scr group .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Caspase activity was detected by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. #P0.05 versus Scr group.


( C , D ) Total caspase 3 were detected by western blot . WT Mfn2 KO subjected or not to XBP ‐ 1 silencing ( Scr , KD ) treated with 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(C, D) Total and cleaved caspase 3 levels were detected by western blot. WT and Mfn2 KO cells subjected or not to XBP‐1 silencing (Scr, and KD) and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( E , F ) Immunodetection of LC3b ‐ I ‐ II in Scr XBP ‐ 1 KD WT or Mfn2 KO treated as indicated ( 1 μM Tg ; 100 nM Baf ) for 6 h . Densitometric is shown in F . Data are mean ± s . e . m . ( n = 3 ) . # P0 . 05 versus Scr group .

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

(E, F) Immunodetection of LC3b‐I and ‐II in Scr and XBP‐1 KD WT or Mfn2 KO cells treated as indicated (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). #P0.05 versus Scr group.


( G ) Mfn2 KO stably expressing mCherry ‐ GFP ‐ LC3b subjected or not to XBP ‐ 1 silencing ( Scr or KD ) were treated with 1 μM Tg , 100 ng / ml Bref , or 0 . 5 μg / ml Tm for 24 h examined by confocal microscopy . Scale bar : 10 μm .

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

(G) Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b and subjected or not to XBP‐1 silencing (Scr or KD) were treated with 1 μM Tg, 100 ng/ml Bref, or 0.5 μg/ml Tm for 24 h and examined by confocal microscopy. Scale bar: 10 μm.


( H ) of mCherry + / GFP ‐ ( mCherry ‐ LC3 ) red per ( a measure of acidic autophagosomes ) is shown . Data are mean ± s . e . m . ( n = 3 ; 100 were analyzed per experiment group ) . # P0 . 05 versus Scr group . Source data for this figure is available on online supplementary information page .

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

(H) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.


( A ) Immunodetection of p ‐ PERK p ‐ eIF2α in Mfn2 KO MEFs , Mfn2 knockdown 3T3 ‐ L1 fibroblasts , Mfn2 knockdown C2C12 myoblasts .

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

(A) Immunodetection of p‐PERK and p‐eIF2α in Mfn2 KO MEFs, Mfn2 knockdown 3T3‐L1 fibroblasts, and Mfn2 knockdown C2C12 myoblasts.


( B ) Densitometric of p ‐ PERK p ‐ eIF2α in MEFs . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT .

O O O O O O O B-MEASURED_VAR 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

(B) Densitometric quantification of p‐PERK and p‐eIF2α in MEFs. Data are mean±s.e.m. (n=3). *P0.05 versus WT cells.


( C , D ) Immunodetection of p ‐ eIF2α , eIF2α , CHOP , XBP ‐ 1s , LC3b , p62 in Mfn2 ‐ deficient liver ( C ) or skeletal muscle ( D ) from tissue ‐ specific KO mice .

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

(C, D) Immunodetection of p‐eIF2α, eIF2α, CHOP, XBP‐1s, LC3b, and p62 in Mfn2‐deficient liver (C) or skeletal muscle (D) from tissue‐specific KO mice.


( E ) Co ‐ immunoprecipitation of Mfn2 PERK in WT or Mfn2 KO stably expressing PERK ‐ myc or an empty vector ( pBABE ‐ myc ; negative control ) .

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

(E) Co‐immunoprecipitation of Mfn2 and PERK in WT or Mfn2 KO cells stably expressing PERK‐myc or an empty vector (pBABE‐myc; negative control).


( F ) Co ‐ immunoprecipitation of endogenous Mfn2 PERK in WT MEF lysates . PERK was immunoprecipitated with a C ‐ terminal antibody co ‐ immunoprecipitation of Mfn2 was detected by western blot . Source data for this figure is available on online supplementary information page .

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

(F) Co‐immunoprecipitation of endogenous Mfn2 and PERK in WT MEF lysates. PERK was immunoprecipitated with a C‐terminal antibody and co‐immunoprecipitation of Mfn2 was detected by western blot.Source data for this figure is available on the online supplementary information page.


( A ) Representative confocal of mitochondrial in scrambled ( Scr ) PERK KD Mfn2 KO stained with MitoTracker Green .

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(A) Representative confocal images of mitochondrial morphology in scrambled (Scr) and PERK KD Mfn2 KO cells stained with MitoTracker Green.


( B ) Mitochondrial . Data are mean ± s . e . m . ( n = 3 ; 100 were analyzed per experiment group ) . * P0 . 05 versus Scr group .

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(B) Mitochondrial morphology quantification. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus Scr group.


( C , D ) Representative confocal of mitochondrial in scrambled ( Scr ) PERK KD WT ( C ) or scrambled ( Scr ) PERK KD Mfn1 KO ( D ) stained with MitoTracker Green .

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(C, D) Representative confocal images of mitochondrial morphology in scrambled (Scr) and PERK KD WT cells (C) or scrambled (Scr) and PERK KD Mfn1 KO cells (D) stained with MitoTracker Green.


( E ) Representative confocal of mitochondrial in Mfn2 KO treated with 1 μM TUDCA or 10 mM 4 ‐ phenyl butyric acid for 6 h stained with MitoTracker Green .

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(E) Representative confocal images of mitochondrial morphology in Mfn2 KO cells treated with 1 μM TUDCA or 10 mM 4‐phenyl butyric acid for 6 h and stained with MitoTracker Green.


( F ) Flow ‐ cytometry of ROS in Scr PERK KD WT Mfn2 KO

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(F) Flow‐cytometry quantification of ROS levels in Scr and PERK KD WT and Mfn2 KO cells


( G ) or Scr PERK KD WT Mfn1 using DHR1 , 2 , 3 . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT group , # P0 . 05 versus Scr group . Scale bars : 10 μm .

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(G) or Scr and PERK KD WT and Mfn1 cells using DHR1,2,3. Data are mean±s.e.m. (n=4). *P0.05 versus WT group, #P0.05 versus Scr group. Scale bars: 10 μm.


( A ) Mitochondrial calcium overload in Scr PERK KD Mfn2 KO . were loaded with Rhod ‐ 2 then treated with 2 . 5 mM CaCl2 ( left ) . Calcium uptake was monitored by confocal microscopy . Representative confocal of mitochondrial in WT Mfn2 KO stained with Rhod ‐ 2 incubated with 2 . 5 mM CaCl2 for 5 min ( right ) . Scale bars : 10 μm .

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(A) Mitochondrial calcium overload in Scr and PERK KD Mfn2 KO cells. Cells were loaded with Rhod‐2 and then treated with 2.5 mM CaCl2 (left). Calcium uptake was monitored by confocal microscopy. Representative confocal images of mitochondrial morphology in WT and Mfn2 KO cells stained with Rhod‐2 and incubated with 2.5 mM CaCl2 for 5 min (right). Scale bars: 10 μm.


( B - E ) Mitochondrial oxygen consumption ( OCR ) was measured in Scr PERK KD Mfn2 KO ( B ) , Scr PERK KD WT ( C ) , Scr PERK KD Mfn1 KO ( D ) , WT stably expressing PERK ‐ myc ( E ) . Data are mean of three independent experiments . following parameters were measured : oxygen consumption under routine conditions ( DMEM with 5 . 5 mM glucose ) , maximal respiratory capacity reached after uncoupling with FCCP , respiratory leak , measured after inhibition of ATP synthase with oligomycin . Data are mean ± s . e . m . * P0 . 05 versus Scr group or versus empty plasmid group .

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(B-E) Mitochondrial oxygen consumption (OCR) was measured in Scr and PERK KD Mfn2 KO cells (B), Scr and PERK KD WT cells (C), Scr and PERK KD Mfn1 KO cells (D), and WT cells stably expressing PERK‐myc (E). Data are mean of three independent experiments. The following parameters were measured: oxygen consumption under routine conditions (DMEM with 5.5 mM glucose), maximal respiratory capacity reached after uncoupling with FCCP, and respiratory leak, measured after inhibition of ATP synthase with oligomycin. Data are mean±s.e.m. *P0.05 versus Scr group or versus the empty plasmid group.


( F , G ) Representative confocal of mitochondrial in WT stably expressing PERK ‐ myc stained with MitoTracker Green . Scale bars : 10 μm . Quantitative of mitochondrial is shown in G . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus empty plasmid group .

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(F, G) Representative confocal images of mitochondrial morphology in WT cells stably expressing PERK‐myc stained with MitoTracker Green. Scale bars: 10 μm. Quantitative analysis of mitochondrial morphology is shown in G. Data are mean±s.e.m. (n=3). *P0.05 versus the empty plasmid group.


A . Vps10yEGFP localization . Yeast carrying Vps10yEGFP expressing indicated vps35 alleles as sole source of Vps35 were logarithmically grown in SC medium . Their vacuoles were labelled with FM4 - 64 . were harvested by brief centrifugation immediately imaged by confocal microscopy . Single confocal planes are shown . A brightfield image was used to outline boundaries ( shown in merged ) . B . Co - localization of Vps10yEGFP FM4 - 64 in from a was measured using Pearson ' s coefficient . 15 Confocal planes with 20 to 30 each from 3 independent experiments were analyzed .

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A. Vps10yEGFP localization. Yeast cells carrying Vps10yEGFP and expressing the indicated vps35 alleles as the sole source of Vps35 were logarithmically grown in SC medium. Their vacuoles were labelled with FM4-64. Cells were harvested by brief centrifugation and immediately imaged by confocal microscopy. Single confocal planes are shown. A brightfield image was used to outline the cell boundaries (shown in the merged images). B. Co-localization of Vps10yEGFP and FM4-64 in cells from a was measured using Pearson's coefficient. 15 Confocal planes with 20 to 30 cells each from 3 independent experiments were analyzed. Means


B . Influence of VPS35 variants on GLUT1 . HK2 silenced for VPS35 were transfected with a plasmid carrying siRNA resistant wildtype or mutant forms of GFP - VPS35 . GLUT1 was detected by fixation immunofluorescence as in a . Maximum projections of image stacks ( step in z of 300 nm ) are shown .

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B. Influence of VPS35 variants on GLUT1. HK2 cells silenced for VPS35 were transfected with a plasmid carrying siRNA resistant wildtype or mutant forms of GFP-VPS35. GLUT1 was detected by fixation and immunofluorescence staining as in a. Maximum projections of image stacks (step size in z of 300 nm) are shown.


C . of GLUT1 immunofluorescence in from B . Regions of interest ( ROIs ) corresponding to expressing indicated VPS35 variants , some regions outside ( background ) , were manually defined using ImageJ software . Total was integrated corrected for background . 105 per condition stemming from 3 independent experiments were analyzed . was performed with 99 % confidence : * * * p < 0 . 001 .

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C. Quantification of GLUT1 immunofluorescence in cells from B. Regions of interest (ROIs) corresponding to cells expressing the indicated VPS35 variants, and some regions outside the cells (background), were manually defined using ImageJ software. Total cell fluorescence was integrated and corrected for background fluorescence. 105 cells per condition stemming from 3 independent experiments were analyzed. The analysis was performed with 99% confidence: *** p < 0.001.


D . Expression of Vps35 variants in from B was analyzed by SDS - PAGE Western blot against Vps35 . Tubulin served as loading control .

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D. Expression of Vps35 variants in cells from B was analyzed by SDS-PAGE and Western blot against Vps35. Tubulin served as loading control.


A PHIP - 1 interaction with GPB - 1 by yeast two - hybrid . reporter PJ69 - 4A was co - transformed with expression vectors encoding GAL4 DBD - PHIP - 1 ( H45A ) GAL4 AD - GPB - 1 , as indicated . Yeast strains carrying indicated plasmids were cultured on a selective plate lacking histidine containing 5 mM 5 - aminotriazole for 4 days .

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A PHIP-1 interaction with GPB-1 by yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-PHIP-1(H45A) and GAL4 AD-GPB-1, as indicated. Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5-aminotriazole for 4 days.


A His - phosphorylation of GPB - 1 in . phip - 1 ( km96 ) mutant carrying 3XFLAG : : gpb - 1 or 3XFLAG : : gpb - 1 ( H266F ) knock - in allele were lysed . lysates were treated with or without heating ( 95 ° C ) immunoblotted ( IB ) with anti - 3 - pHis anti - FLAG antibodies .

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A His-phosphorylation of GPB-1 in animals. The phip-1(km96) mutant animals carrying the 3XFLAG::gpb-1 or 3XFLAG::gpb-1(H266F) knock-in allele were lysed. The lysates were treated with or without heating (95°C) and immunoblotted (IB) with anti-3-pHis and anti-FLAG antibodies.


B NDK - 1 phosphorylates GPB - 1 in vitro . COS - 7 were co - transfected with HA - GPB - 1 or HA - GPB - 1 ( H266F ) T7 - GPC - 2 , lysates were immunoprecipitated ( IP ) with anti - HA antibodies . Immunopurified GPB - 1 was subjected to in vitro kinase with recombinant GST - NDK - 1 . Phosphorylated GPB - 1 was detected by immunoblotting ( IB ) with anti - 3 - pHis antibodies .

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B NDK-1 phosphorylates GPB-1 in vitro. COS-7 cells were co-transfected with HA-GPB-1 or HA-GPB-1(H266F) and T7-GPC-2, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies. Immunopurified GPB-1 was subjected to the in vitro kinase assay with recombinant GST-NDK-1. Phosphorylated GPB-1 was detected by immunoblotting (IB) with anti-3-pHis antibodies.


C PHIP - 1 dephosphorylates GPB - 1 in vitro . COS - 7 were co - transfected with HA - GPB - 1 T7 - GPC - 2 , lysates were immunoprecipitated ( IP ) with anti - HA antibodies . immunopurified HA - GPB - 1 was first subjected to in vitro kinase with recombinant GST - NDK - 1 . Phosphorylated GPB - 1 was then equally aliquoted subjected to in vitro phosphatase with recombinant GST - PHIP - 1 or its variants . Phosphorylated GPB - 1 was detected by immunoblotting ( IB ) with anti - 3 - pHis antibodies .

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C PHIP-1 dephosphorylates GPB-1 in vitro. COS-7 cells were co-transfected with HA-GPB-1 and T7-GPC-2, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies. The immunopurified HA-GPB-1 was first subjected to the in vitro kinase assay with recombinant GST-NDK-1. Phosphorylated GPB-1 was then equally aliquoted and subjected to the in vitro phosphatase assay with recombinant GST-PHIP-1 or its variants. Phosphorylated GPB-1 was detected by immunoblotting (IB) with anti-3-pHis antibodies.


A PHIP - 1 interaction with UNC - 51 by yeast two - hybrid . reporter PJ69 - 4A was co - transformed with expression vectors encoding GAL4 DBD - PHIP - 1 ( H45A ) GAL4 AD - UNC - 51 ( 274 - 856 ) , as indicated . Yeast strains carrying indicated plasmids were cultured on a selective plate lacking histidine containing 5 mM 5 - aminotriazole for 4 days .

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A PHIP-1 interaction with UNC-51 by yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-PHIP-1(H45A) and GAL4 AD-UNC-51(274-856), as indicated. Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5-aminotriazole for 4 days.


C UNC - 51 phosphorylates PHIP - 1 at Ser - 112 . COS - 7 were co - transfected with Flag - PHIP - 1 ( WT or mutants ) GFP - UNC - 51 [ WT or ∆ AIKAI ( KD ) ] , lysates were analyzed using Phos - tag SDS - PAGE . Total lysates were immunoblotted ( IB ) with antibodies , as indicated . Filled open arrowheads indicate unmodified phosphorylated PHIP - 1 , respectively . Asterisk indicates non - specific band .

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C UNC-51 phosphorylates PHIP-1 at Ser-112. COS-7 cells were co-transfected with Flag-PHIP-1 (WT or mutants) and GFP-UNC-51 [WT or ∆AIKAI (KD)], and cell lysates were analyzed using Phos-tag SDS-PAGE. Total lysates were immunoblotted (IB) with antibodies, as indicated. Filled and open arrowheads indicate unmodified and phosphorylated PHIP-1, respectively. Asterisk indicates non-specific band.


A . Western blot of Tace using lysates from WT TaceFl / Fl ; P0 - Cre mousesciatic nerves at P10 ( 65ug of total lysate loaded ) . arrowhead indicates pro - arrow shows active form of Tace . Representative of two independent experiments .

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A. Western blot analysis of Tace using lysates from WT and TaceFl/Fl;P0-Cre mousesciatic nerves at P10 (65ug of total protein lysate loaded). The arrowhead indicates the pro-protein and the arrow shows the cleaved active form of Tace. Representative of two independent experiments.


B . Western blot ( 50ug of total lysate loaded ) shows that Tace expression in axons is maximal at P5 - P10 then progressively declines in adult . Representative of two independent experiments .

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B. Western blot analysis (50ug of total protein lysate loaded) shows that Tace expression in axons is maximal at P5-P10 and then progressively declines in the adult. Representative of two independent experiments.


C . Western blot of Tace in Mtmr2 - / - sciatic nerves at P5 , P10 , P20 . Representative of two independent experiments .

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C. Western blot analysis of Tace in Mtmr2-/- sciatic nerves at P5, P10, and P20. Representative of two independent experiments.


D . Western blot of Tace using lysates from Mtmr2 - / - mouse sciatic nerves at 5 months , with , p = 0 . 10 , two - Mann Whitney U . Representative of two independent experiments .

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D. Western blot analysis of Tace using lysates from Mtmr2-/- mouse sciatic nerves at 5 months, with quantification, p=0.10, two-tailed Mann Whitney U test. Representative of two independent experiments.


E . Phosphorylation of ErbB2 in Mtmr2 - / - sciatic nerves at P2 , P10 P15 . At P2 , each lane is a pool of n = 7 per genotype , representative of six independent experiments using n = 6 different nerve pools per genotype , using WT values arbitrary set to1 , p = 0 . 0313 , Wilcoxon Rank Sum . At P10 P15 , mean values of two samples are shown . At P10 , each lane is a pool of n = 3 / 4 per genotype , representative of three independent experiments using n = 6 different pools per genotype . Nf - l is neurofilament chain Clx is calnexin .

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E. Phosphorylation of ErbB2 in Mtmr2-/- sciatic nerves at P2, P10 and P15. At P2, each lane is a pool of n=7 animals per genotype, representative of six independent experiments using n=6 different nerve pools per genotype, and quantification using WT values arbitrary set to1, p=0.0313, Wilcoxon Rank Sum test. At P10 and P15, mean values of two samples are shown. At P10, each lane is a pool of n=3/4 animals per genotype, representative of three independent experiments using n=6 different pools per genotype. Nf-l is the neurofilament light chain and Clx is calnexin.


F . Phosphorylation of ErbB2 in Mtmr2 - / - Schwann / DRG neuron co - cultures after 4 days of ascorbic acid treatment . Each lane is a pool from at least 10 coverslips / DRG per genotype . Representative of two independent experiments using n = 4 different pools of coverslips / DRG per genotype , p = 0 . 20 , two - Mann Whitney U .

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F. Phosphorylation of ErbB2 in Mtmr2-/- Schwann cell/DRG neuron co-cultures after 4 days of ascorbic acid treatment. Each lane is a pool from at least 10 coverslips/DRG per genotype. Representative of two independent experiments using n=4 different pools of coverslips/DRG per genotype, p=0.20, two-tailed Mann Whitney U test.


A . ShRNA targeting Nrg1 type III were validated in rat purified neurons . Western blot for Nrg1 shows reduction of its expression ( pro - at 150 KDa ) using two different harpins , representative of two independent experiments . Clx is calnexin .

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A. ShRNA targeting Nrg1 type III were validated in rat purified neurons. Western blot analysis for Nrg1 shows reduction of its expression (pro-protein at 150 KDa) using two different harpins, representative of two independent experiments. Clx is calnexin.


B . ShRNA # 1 2 downregulate Nrg1 type III expression also in myelin - forming Schwann / DRG neuron co - cultures analyzed after 7 days of ascorbic acid treatment , as shown by western blot , representative of two independent experiments . Clx is calnexin .

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B. ShRNA#1 and 2 downregulate Nrg1 type III expression also in myelin-forming Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, as shown by western blot analysis, representative of two independent experiments. Clx is calnexin.


C , D . Titration of shRNA # 1 ( C ) of shRNA # 2 ( D ) LVs ( lentiviral vectors ) on WT Schwann / DRG neuron co - cultures analyzed after 7 days of ascorbic acid treatment , with of mean number of Mbp positive segments Schwann number . In ( C ) n = 20 ( SCR , Scramble ) , n = 11 ( LV # sh1 , 12 . 5 % ) , n = 16 ( LV # sh1 , 25 % ) , n = 9 ( LV # sh1 , 50 % ) DRG / coverslips from two independent experiments . Results are mean ± , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc . In ( D ) n = 14 ( SCR ) , n = 10 ( LV # sh2 , 12 . 5 % ) , n = 12 ( LV # sh2 , 25 % ) , n = 79 ( LV # sh2 , 50 % ) DRG / coverslips , representative of two independent experiments . Results are mean ± , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc .

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C, D. Titration of shRNA #1 (C) and of shRNA #2 (D) LVs (lentiviral vectors) on WT Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, with quantification of the mean number of Mbp positive segments and Schwann cell number. In (C) n= 20 (SCR, Scramble), n= 11 (LV #sh1, 12.5%), n= 16 (LV #sh1, 25%), n= 9 (LV #sh1, 50%) DRG/coverslips from two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test. In (D) n= 14 (SCR), n= 10 (LV #sh2, 12.5%), n= 12 (LV #sh2, 25%), n= 79 (LV #sh2, 50%) DRG/ coverslips, representative of two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


E . Western blot of Nrg1 Akt phosphorylation ( S473 ) shows that activation of Akt decreases following Nrg1 type III downregulation ( p - Akt / Akt / Actin , between Mtmr2 - / - Scramble - treated Mtmr2 - / - sh # 1 - treated using 12 . 5 % LV is 1 : 0 . 85 ) . Each lane is a pool of at least 10 different DRG / coverslips , representative of two independent experiments . Bar in ( C , D ) is 100 μm ; in ( E ) is 20 μm .

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E. Western blot analysis of Nrg1 and Akt phosphorylation (S473) shows that activation of Akt decreases following Nrg1 type III downregulation (p-Akt/Akt/Actin, ratio between Mtmr2-/- Scramble-treated and Mtmr2-/- sh#1-treated using 12.5% LV is 1 : 0.85). Each lane is a pool of at least 10 different DRG/coverslips, representative of two independent experiments. Bar in (C, D) is 100 μm; in (E) is 20 μm.


E . Representative confocal of Mtmr2 - / - co - cultures transduced using LV expressing Nrg1 type III shRNA # 1 , with of of myelin outfoldings , n = number of DRG / coverslips , n = 9 , 382 ( SCR ) , n = 9 , 368 ( LV # sh1 , 12 . 5 % ) , n = 8 , 327 ( LV # sh1 , 25 % ) . Asterisks indicate with myelin outfoldings . Results are mean ± , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc , representative of two independent experiments .

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E. Representative confocal images of Mtmr2-/- co-cultures transduced using LV expressing Nrg1 type III shRNA #1, with quantification of the percentage of myelin outfoldings, n= number of DRG/coverslips, n= 9, 382 fibers (SCR), n=9, 368 fibers (LV #sh1, 12.5%), n= 8, 327 fibers (LV #sh1, 25%). Asterisks indicate fibers with myelin outfoldings. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test, representative of two independent experiments.


A . Titration of rhTACE on wild - type ( WT ) co - cultures stained for Mbp with of Mbp positive Schwann nuclei : n = 18 ( NT , not treated ) , n = 6 ( 5ng / ml ) , n = 20 ( 10ng / ml ) , n = 7 ( 50ng / ml ) DRGs / covers from two independent experiments , p = 0 . 0053 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc .

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

A. Titration of rhTACE on wild-type (WT) co-cultures stained for Mbp with quantification of Mbp positive fibers and Schwann cell nuclei: n=18 (NT, not treated), n=6 (5ng/ml), n=20 (10ng/ml), n=7 (50ng/ml) DRGs/covers from two independent experiments, p=0.0053, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


B . Western blot of lysates from Mtmr2 - / - cultures treated using 10ng / ml rhTACE showing Nrg1 expression Akt activation ( S473 phosphorylation ) . Each lane is a lysate of at least n = 10 DRGs / coverslips . In Mtmr2 - / - ( 10ng / ml rhTACE ) as compared to Mtmr2 - / - ( NT ) , Nrg1 ( 150 KDa ) / actin is 0 . 8 : 1 ; Nrg1 ( 65 KDa ) / actin is 0 . 48 : 1 ; p - Akt / Akt / actin is 0 . 6 : 1 .

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B. Western blot analysis of lysates from Mtmr2-/- cultures treated using 10ng/ml rhTACE showing Nrg1 expression and Akt activation (S473 phosphorylation). Each lane is a lysate of at least n=10 DRGs/coverslips. In Mtmr2-/- (10ng/ml rhTACE) as compared to Mtmr2-/- (NT), Nrg1(150 KDa)/actin ratio is 0.8:1; Nrg1(65 KDa)/actin ratio is 0.48:1; and p-Akt/Akt/actin ratio is 0.6:1.


B . Immunohistochemistry confocal microscopy of Mtmr2 - / - cultures treated with 5ng / ml 10ng / ml rhTACE , with of of myelin outfoldings ; n = 10 ( NT ) , n = 10 ( 5ng / ml ) , n = 11 ( 10ng / ml ) , DRGs / coverslips . A total of 547 , 557 , 571 Mbp were quantified , respectively , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc .

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

B. Immunohistochemistry and confocal microscopy of Mtmr2-/- cultures treated with 5ng/ml and 10ng/ml rhTACE, with quantification of the percentage of myelin outfoldings; n=10 (NT), n=10 (5ng/ml), n=11 (10ng/ml), DRGs/coverslips. A total of 547, 557, and 571 Mbp fibers were quantified, respectively, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


C . Titration of WT co - cultures using different amounts of Niacin , nicotinic acid , with of Mbp positive Schwann cellnuclei , n = 9 ( NT ) , n = 8 ( 1mM ) , n = 9 ( 5mM ) , n = 8 ( 10mM ) , DRGs / coverslips . Representative of three independent experiments , p = 0 . 0055 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc .

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

C. Titration of WT co-cultures using different amounts of Niacin, nicotinic acid, with quantification of Mbp positive fibers and Schwann cellnuclei, n=9 (NT), n=8 (1mM), n=9 (5mM), n=8 (10mM), DRGs/ coverslips. Representative of three independent experiments, p=0.0055, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


D . Immunohistochemistry confocal microscopy of Mtmr2 - / - cultures treated with 1 mM 5mM Niacin , with of of myelin outfoldings ; n = 31 ( NT ) , n = 14 ( 1mM ) , n = 29 ( 5mM ) DRGs / coverslips from two independent experiments . A total of 1256 , 489 , 1305 , respectively , were quantified , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc .

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

D. Immunohistochemistry and confocal microscopy of Mtmr2-/- cultures treated with 1 mM and 5mM Niacin, with quantification of the percentage of myelin outfoldings; n=31 (NT), n=14 (1mM), and n=29 (5mM) DRGs/coverslips from two independent experiments. A total of 1256, 489, and 1305 fibers, respectively, were quantified, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


D . Tace activity was measured from lysates of Niacin - treated cultures , n = 7 ( KO NT ) n = 8 ( KO 5mM Niacin ) number of independent plates , each containing from 10 - 15 DRGs plated . Note that Tace activity was similar between WT Mtmr2 - / - cultures , one - Mann Whitney U , p = 0 . 037 .

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D. Tace activity was measured from lysates of Niacin-treated cultures, n=7 (KO NT) and n=8 (KO 5mM Niacin) number of independent plates, each containing from 10-15 DRGs plated. Note that Tace activity was similar between WT and Mtmr2-/- cultures, one-tailed Mann Whitney U test, p=0.037.


E . Niacin treatment of Tace - / - explants does not rescue hypermyelination , with , n = 8 DRGs / coverslips per condition , p = 0 . 0056 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc . Representative of three independent experiments .

O O B-CONTROLLED_VAR 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

E. Niacin treatment of Tace-/- explants does not rescue hypermyelination, with quantification, n=8 DRGs/coverslips per condition, p=0.0056, nonparametric one-way ANOVA, followed by Dunn's post hoc test. Representative of three independent experiments.


A . Representative of semithin section of sciatic nerves from Mtmr2 - / - Mtmr2 - / - ; Nrg1 ( III ) + / - mice at 6 months . Myelin outfoldings ( arrowheads ) are reduced in Mtmr2 - / - ; Nrg1 ( III ) + / - nerves ( n = 9 ) as compared to Mtmr2 - / - ( n = 8 ) , results are mean ± , p = 0 . 0037 , two - Mann Whitney U . B . G - on sciatic nerves from Mtmr2 - / - , Mtmr2 - / - ; Nrg1 ( III ) + / - , Nrg1 ( III ) + / - mice at 6 months indicates that myelin thickness of Mtmr2 - / - ; Nrg1 ( III ) + / - is similar to Nrg1 ( III ) + / - nerves different from Mtmr2 - / - , which is in normal range as already reported ( Bonneick et al , 2005 ) , n = 5 per genotype . Mean g - ratios : Mtmr2 - / - 0 . 67 ± 0 . 03 , 2129 , n = 5 ; Mtmr2 - / - ; Nrg1 ( III ) + / - 0 . 71 ± 0 . 03 , 2139 , n = 5 ; Nrg1 ( III ) + / - 0 . 72 ± 0 . 03 , 2152 , n = 4 . Mtmr2 - / - as compared to Mtmr2 - / - ; Nrg1 ( III ) + / - , p = 0 . 0145 ; Mtmr2 - / - as compared to Nrg1 ( III ) + / - , p = 0 . 0030 , Mtmr2 - / - ; Nrg1 ( III ) + / - as compared to Nrg1 ( III ) + / - p = 0 . 3193 , repeated measures ANOVA .

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

A. Representative images of semithin section analysis of sciatic nerves from Mtmr2-/- and Mtmr2-/-;Nrg1 (III)+/- mice at 6 months. Myelin outfoldings (arrowheads) are reduced in Mtmr2-/-;Nrg1 (III)+/- nerves (n=9) as compared to Mtmr2-/- (n=8), results are mean ± SEM, p=0.0037, two-tailed Mann Whitney U test.B. G-ratio analysis on sciatic nerves from Mtmr2-/-, Mtmr2-/-;Nrg1 (III)+/-, and Nrg1 (III)+/- mice at 6 months indicates that myelin thickness of Mtmr2-/-;Nrg1 (III)+/- is similar to Nrg1 (III)+/- nerves and different from Mtmr2-/-, which is in the normal range as already reported (Bonneick et al, 2005), n=5 animals per genotype.Mean g-ratios: Mtmr2-/- 0.67 ± 0.03, 2129 fibers, n=5 animals; Mtmr2-/-;Nrg1 (III)+/- 0.71 ± 0.03, 2139 fibers, n=5 animals; Nrg1 (III)+/- 0.72 ± 0.03, 2152 fibers, n=4 animals. Mtmr2-/- as compared to Mtmr2-/-;Nrg1 (III)+/-, p=0.0145; Mtmr2-/- as compared to Nrg1 (III)+/-, p=0.0030, and Mtmr2-/-;Nrg1 (III)+/- as compared to Nrg1 (III)+/- p=0.3193, repeated measures ANOVA.


C . Western blot on lysates from Mtmr2 - / - , Mtmr2 - / - ; Nrg1 ( III ) + / - , Nrg1 ( III ) + / - sciatic nerves at P10 indicates that Akt activation ( S473 phosphorylation ) is similar between Mtmr2 - / - ; Nrg1 ( III ) + / - Nrg1 ( III ) + / - consistent with g - findings , representative of three independent experiments . Phosphorylation of Akt is relative to total Akt normalized on calnexin , Clx .

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C. Western blot analysis on lysates from Mtmr2-/-, Mtmr2-/-;Nrg1 (III)+/-, and Nrg1 (III)+/- sciatic nerves at P10 indicates that Akt activation (S473 phosphorylation) is similar between Mtmr2-/-;Nrg1 (III)+/- and Nrg1 (III)+/- consistent with g-ratio findings, representative of three independent experiments. Phosphorylation of Akt is relative to total Akt normalized on calnexin, Clx.


A . Western blot on lysates from treated not treated explants ( at least 10 DRGs / coverslips per lane ) shows that increased Akt activation ( S473 phosphorylation ) in Vim - / - explants is restored to normal following 5 mM Niacin treatment .

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

A. Western blot analysis on lysates from treated and not treated explants (at least 10 DRGs/ coverslips per lane) shows that increased Akt activation (S473 phosphorylation) in Vim-/- explants is restored to normal levels following 5 mM Niacin treatment.


A . Niacin treatment of Vim - / - explants rescues hypermyelination , n = 10 DRGs / coverslips per condition , with , representative of three independent experiments , p = 0 . 0048 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc .

O O B-CONTROLLED_VAR 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

A. Niacin treatment of Vim-/- explants rescues hypermyelination, n=10 DRGs/ coverslips per condition, with quantification, representative of three independent experiments, p=0.0048, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


B . Niaspan treatment of Vim - / - mice does not affect mouse growth , as growth rate in WT ( n = 8 , saline ; n = 6 Niaspan ) Vim - / - ( n = 10 saline n = 10 Niaspan ) mice either saline or Niaspan - treated is not significantly different , Linear Mixed Effect ( LME ) models .

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B. Niaspan treatment of Vim-/- mice does not affect mouse growth, as the growth rate in WT (n=8, saline; n=6 Niaspan) and Vim-/- (n=10 saline and n=10 Niaspan) mice either saline or Niaspan-treated is not significantly different, Linear Mixed Effect (LME) models.


B . Niaspan administration ( daily i . p . injection of 160mg / Kg Niaspan starting at P15 for 15 days ) enhances Tace activity in Vim - / - nerves at P30 , n = 6 per genotype , p = 0 . 0325 , one - Mann Whitney U .

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B. Niaspan administration (daily i.p. injection of 160mg/Kg Niaspan starting at P15 for 15 days) enhances Tace activity in Vim-/- nerves at P30, n=6 animals per genotype, p=0.0325, one-tailed Mann Whitney U test.


C . Semithin section g - analyses of sciatic nerves at P30 shows that Niaspan does not alter myelin thickness in WT nerves , whereas restores myelin thickness to normal values in Vim - / - nerves . G - values : WT saline , 0 . 71 ± 0 . 003 , 1872 , n = 5 ; WT Niaspan , 0 . 71 ± 0 . 004 , 1882 , n = 5 ; Vim - / - saline 0 . 69 ± 0 . 004 , 2313 , n = 6 ; Vim - / - Niaspan 0 . 72 ± 0 . 01 , 1616 , n = 5 . WT saline as compared to Vim - / - saline , p = 0 . 0588 ; Vim - / - saline as compared to Vim - / - Niaspan , p = 0 . 0431 , repeated measures ANOVA , representative of two independent experiments . number of myelinated is similar between 4 groups as shown , p = 0 . 3042 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc .

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 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 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

C. Semithin section and g-ratio analyses of sciatic nerves at P30 shows that Niaspan does not alter myelin thickness in WT nerves, whereas restores myelin thickness to normal values in Vim-/- nerves. G-ratio values: WT saline, 0.71 ± 0.003, 1872 fibers, n= 5 animals; WT Niaspan, 0.71 ± 0.004, 1882 fibers, n= 5 animals; Vim-/- saline 0.69 ± 0.004, 2313 fibers, n= 6 animals; Vim-/- Niaspan 0.72 ± 0.01, 1616 fibers, n=5 animals. WT saline as compared to Vim-/- saline, p=0.0588; Vim-/- saline as compared to Vim-/- Niaspan, p=0.0431, repeated measures ANOVA, representative of two independent experiments. The number of myelinated fibers is similar between the 4 groups as shown, p=0.3042, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


A . Niaspan administration ( daily i . p . injection of 160mg / Kg starting at P15 for 60 days ) does not affect growth of Mtmr2 - / - mice . growth rates of Mtmr2 - / - WT both saline - treated are significantly different , p < 0 . 0001 , Linear Mixed Effect ( LME ) models , as already reported ( Bolino et al , 2004 ) , n = 8 per condition .

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A. Niaspan administration (daily i.p. injection of 160mg/Kg starting at P15 for 60 days) does not affect the growth of Mtmr2-/- mice. The growth rates of Mtmr2-/- and WT both saline-treated are significantly different, p<0.0001, Linear Mixed Effect (LME) models, as already reported (Bolino et al, 2004), n=8 animals per condition.


B . Niaspan administration reduces of myelin outfoldings ( red asterisks ) in Mtmr2 - / - sciatic nerves without affecting number of myelinated as assessed by semithin section at P75 , n = 8 mice per genotype , results are mean ± , p = 0 . 0093 , two - Mann Whitney U , representative of three independent experiments . Niaspan treatment does not alter myelin thickness in WT or Mtmr2 - / - mice as assessed by g - ( WT saline - treated , 0 . 67 ± 0 . 003 , 2014 ; Mtmr2 - / - Niaspan - treated , 0 . 67 ± 0 . 007 , 1924 ; Mtmr2 - / - saline - treated , 0 . 68 ± 0 . 003 , 2244 ; n = 4 per condition ) .

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

B. Niaspan administration reduces the percentage of myelin outfoldings (red asterisks) in Mtmr2-/- sciatic nerves without affecting the number of myelinated fibers as assessed by semithin section analysis at P75, n= 8 mice per genotype, results are mean ± SEM, p=0.0093, two-tailed Mann Whitney U test, representative of three independent experiments. Niaspan treatment does not alter myelin thickness in WT or Mtmr2-/- mice as assessed by g-ratio analysis (WT saline-treated, 0.67 ± 0.003, 2014 fibers; Mtmr2-/- Niaspan-treated, 0.67 ± 0.007, 1924 fibers; Mtmr2-/- saline-treated, 0.68 ± 0.003, 2244 fibers; n=4 animals per condition).


A . Western blot of lysates from Pmp22 + / - WT sciatic nerves at P10 shows that Akt phosphorylation ( S473 ) is increased in Pmp22 + / - nerves , results are mean ± , p = 0 . 0317 , two - Mann Whitney U , representative of two independent experiments .

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

A. Western blot analysis of lysates from Pmp22+/- and WT sciatic nerves at P10 shows that Akt phosphorylation (S473) is increased in Pmp22+/- nerves, results are mean ± SEM, p=0.0317, two-tailed Mann Whitney U test, representative of two independent experiments.


B . Tace activity as measured in Pmp22 + / - WT sciatic nerves at P30 ( n = 6 mice per genotype , results are mean ± , p = 0 . 0070 , two - Mann Whitney U ) .

O O B-MEASURED_VAR 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

B. Tace activity as measured in Pmp22+/- and WT sciatic nerves at P30 (n=6 mice per genotype, results are mean ± SEM, p=0.0070, two-tailed Mann Whitney U test).


B . Western blot of Tace using lysates from Pmp22 + / - WT sciatic nerves at different stages of development . arrowhead indicates pro - arrow shows active form of Tace . Representative of two independent experiments .

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B. Western blot analysis of Tace using lysates from Pmp22+/- and WT sciatic nerves at different stages of development. The arrowhead indicates the pro-protein and the arrow shows the cleaved active form of Tace. Representative of two independent experiments.


C . Representative of semithin section of sciatic nerves at P45 from Pmp22 + / - saline Pmp22 + / - Niaspan - treated mice . Niaspan was administered daily by i . p . injection at 160mg / Kg starting at P15 for 30 days . C ' . of tomacula in sciatic nerves was assessed by ultrastructural , results are mean ± , n = 6 per condition , p = 0 . 0022 , two - Mann Whitney U , representative of two independent experiments . D . G - on semithin sections of sciatic nerves shows that increased myelin thickness in Pmp22 + / - sciatic nerves at P45 is restored to normal values following Niaspan treatment . Mean g - values , WT saline - treated : 0 . 69 ± 0 . 002 , 2052 , n = 5 ; Pmp22 + / - saline - treated : 0 . 66 ± 0 . 003 , 2643 , n = 6 ; Pmp22 + / - Niaspan - treated , 0 . 68 ± 0 . 002 , 2311 , n = 6 . WT saline as compared to Pmp22 + / - saline - treated , p < 0 . 0001 ; Pmp22 + / - saline - treated as compared to Pmp22 + / - Niaspan - treated , p = 0 . 0002 ; Pmp22 + / - Niaspan - treated as compared to WT saline , p = 0 . 0899 ; repeated measures ANOVA . E . number of myelinated is similar between 3 groups analyzed , p = 0 . 4362 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc .

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C. Representative images of semithin section analysis of sciatic nerves at P45 from Pmp22+/- saline and Pmp22+/- Niaspan-treated mice. Niaspan was administered daily by i.p. injection at 160mg/Kg starting at P15 for 30 days.C'. The percentage of tomacula in sciatic nerves was assessed by ultrastructural analysis, results are mean ± SEM, n= 6 animals per condition, p=0.0022, two-tailed Mann Whitney U test, representative of two independent experiments.D. G-ratio analysis on semithin sections of sciatic nerves shows that increased myelin thickness in Pmp22+/- sciatic nerves at P45 is restored to normal values following Niaspan treatment. Mean g-ratio values, WT saline-treated: 0.69 ± 0.002, 2052 fibers, n=5 animals; Pmp22+/- saline-treated: 0.66 ± 0.003, 2643 fibers, n=6 animals; Pmp22+/- Niaspan-treated, 0.68 ± 0.002, 2311 fibers, n=6 animals. WT saline as compared to Pmp22+/- saline-treated, p<0.0001; Pmp22+/- saline-treated as compared to Pmp22+/- Niaspan-treated, p=0.0002; Pmp22+/- Niaspan-treated as compared to WT saline, p=0.0899; repeated measures ANOVA.E. The number of myelinated fibers is similar between the 3 groups analyzed, p=0.4362, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


A . Western blot to detect phosphorylation of Akt ( S473 ) on lysates from Pmp22 - / - WT mice at P20 , with . Results are mean ± , p = 0 . 6857 , two - Mann Whitney U , representative of three independent experiments .

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A. Western blot analysis to detect phosphorylation levels of Akt (S473) on lysates from Pmp22-/- and WT mice at P20, with quantification. Results are mean ± SEM, p=0.6857, two-tailed Mann Whitney U test, representative of three independent experiments.


B . Western blot for Mbp P0 on lysates from Pmp22 - / - WT mice P20 shows decreased myelin expression at both time points in Pmp22 - / - nerves , results are mean ± , p = 0 . 0500 with exception of P0 level at P20 , for which difference between Pmp22 - / - WT nerves is not statistically significant , p = 0 . 1000 , one - Mann Whitney U , representative of two independent experiments .

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B. Western blot analysis for Mbp and P0 on lysates from Pmp22-/- and WT mice P20 shows decreased myelin protein expression levels at both time points in Pmp22-/- nerves, results are mean ± SEM, p=0.0500 with the exception of the P0 protein level at P20, for which the difference between Pmp22-/- and WT nerves is not statistically significant, p=0.1000, one-tailed Mann Whitney U test, representative of two independent experiments.