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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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- 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 cells ) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology . Scale bar : 1 μm .

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

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

O O 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) 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 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 .

O O O 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 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

(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 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 .

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

(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 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 .

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 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

(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 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 .

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 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 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 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 .

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

(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 ) 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 .

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 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 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 .

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

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

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-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

(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 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 .

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

(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 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 .

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 B-MEASURED_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 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

(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 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 .

O O O O O B-CONTROLLED_VAR 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

(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 ) 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 .

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

(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 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 .

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

(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 and Mfn2 KO cells in basal conditions .

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

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 O O O O O O O O O O O O O O O O O O O O O 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 , and XBP ‐ 1s in WT and Mfn2 KO cells treated with 1 μM Tg for the 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 O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O 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 , and ATF4 in WT and Mfn2 KO cells treated with 0 . 5 μg / ml Tunicamycin ( Tm ) for the times indicated .

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

(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 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 .

O O O O O O O B-MEASURED_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 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 and Mfn2 KO cells stably expressing a scrambled shRNA ( Scr ) or a shRNA directed against PERK ( PERK KD ) .

O O O O O B-MEASURED_VAR 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 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 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 .

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 O O O 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 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 .

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 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 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 .

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(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 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 .

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(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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 and p ‐ eIF2α in Mfn2 KO MEFs , Mfn2 knockdown 3T3 ‐ L1 fibroblasts , and Mfn2 knockdown C2C12 myoblasts .

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


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

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(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 , and p62 in Mfn2 ‐ deficient liver ( C ) or skeletal muscle ( D ) from tissue ‐ specific KO mice .

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

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(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 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 .

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(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 images of mitochondrial morphology in scrambled ( Scr ) and PERK KD Mfn2 KO cells 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 morphology quantification . Data are mean ± s . e . m . ( n = 3 ; 100 cells were analyzed per experiment and 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 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 .

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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 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 .

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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 quantification of ROS levels in Scr and PERK KD WT and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 cells from B was analyzed by SDS - PAGE and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 analysis of Tace in Mtmr2 - / - sciatic nerves at P5 , P10 , and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 quantification , representative of three independent experiments , p = 0 . 0048 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test .

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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 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 .

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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 animals per genotype , p = 0 . 0325 , one - tailed Mann Whitney U test .

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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 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 .

O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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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 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 ) .

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 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 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 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 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 .

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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 + / - and WT sciatic nerves at P30 ( n = 6 mice per genotype , results are mean ± SEM , p = 0 . 0070 , two - tailed Mann Whitney U test ) .

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 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 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 .

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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 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 .

O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O 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. 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 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 .

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 O O O 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 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 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 .

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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.


C . Representative images of semithin section analysis of WT and Pmp22 - / - sciatic nerves at P30 . Tomacula are abundant in Pmp22 - / - nerves at this age , where both fibers with thicker myelin ( in the range of < 3 - 4 μm in diameter ) and thinner myelin ( with diameters greater than 4 μm , particularly in motor fasicles ) can be observed . C ' . Niaspan does not ameliorate the phenotype in Pmp22 - / - mice , as indicated by the number of tomacula ( quantification of the percentage of tomacula in the entire sciatic nerve section , results are mean ± SEM , p = 0 . 7400 , Mann Whitney U test , n = 15 animals per condition ) .

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C. Representative images of semithin section analysis of WT and Pmp22-/- sciatic nerves at P30. Tomacula are abundant in Pmp22-/- nerves at this age, where both fibers with thicker myelin (in the range of <3-4 μm in diameter) and thinner myelin (with diameters greater than 4 μm, particularly in motor fasicles) can be observed.C'. Niaspan does not ameliorate the phenotype in Pmp22-/- mice, as indicated by the number of tomacula (quantification of the percentage of tomacula in the entire sciatic nerve section, results are mean ± SEM, p=0.7400, Mann Whitney U test, n= 15 animals per condition).


D . Growth curve of Pmp22 - / - mice treated with Niaspan administered by i . p . injection daily at 160mg / Kg and starting at P15 for 15 days . A significant time by group effect is noted between WT saline and Pmp22 - / - saline ( group effect p = 0 . 9907 and time effect p = 0 . 0057 ) , indicating that the two groups start with similar weight values but then the WT saline group ( n = 6 animals ) grows more in time . When Pmp22 - / - saline ( n = 5 ) and Pmp22 - / - Niaspan - treated ( n = 6 ) are compared , the group effect is statistically significant ( p = 0 . 0076 ) while the time effect is not ( p = 0 . 6677 ) , suggesting that these two groups have a similar growth trend but Pmp22 - / - Niaspan - treated group starts with a higher baseline weight .

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D. Growth curve of Pmp22-/- mice treated with Niaspan administered by i.p. injection daily at 160mg/Kg and starting at P15 for 15 days. A significant time by group effect is noted between WT saline and Pmp22-/- saline (group effect p=0.9907 and time effect p=0.0057), indicating that the two groups start with similar weight values but then the WT saline group (n=6 animals) grows more in time. When Pmp22-/- saline (n=5) and Pmp22-/- Niaspan-treated (n=6) are compared, the group effect is statistically significant (p=0.0076) while the time effect is not (p=0.6677), suggesting that these two groups have a similar growth trend but Pmp22-/- Niaspan-treated group starts with a higher baseline weight.


E . Niaspan does not ameliorate myelin thickness as indicated by g - ratio analysis , with axonal diameter distribution : WT 0 . 68 ± 0 . 003 , 2945 fibers , n = 5 animals ; Pmp22 - / - 0 . 64 ± 0 . 003 , 1557 fibers , n = 6 animals ; Pmp22 - / - Niaspan 0 . 63 ± 0 . 003 , 1445 fibers , n = 6 animals , results are mean ± SEM . WT saline as compared to Pmp22 - / - saline - treated p < 0 . 0001 ; WT saline as compared to Pmp22 - / - Niaspan - treated p < 0 . 0001 , repeated measures ANOVA . Note the loss of myelinated fibers in Pmp22 - / - nerves for all caliber axons .

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E. Niaspan does not ameliorate myelin thickness as indicated by g-ratio analysis, with axonal diameter distribution: WT 0.68 ± 0.003, 2945 fibers, n=5 animals; Pmp22-/- 0.64 ± 0.003, 1557 fibers, n= 6 animals; Pmp22-/- Niaspan 0.63 ± 0.003, 1445 fibers, n=6 animals, results are mean ± SEM. WT saline as compared to Pmp22-/- saline-treated p<0.0001; WT saline as compared to Pmp22-/- Niaspan-treated p<0.0001, repeated measures ANOVA. Note the loss of myelinated fibers in Pmp22-/- nerves for all caliber axons.


Dynamics of histone modifications after an acclimatizing HS ( ACC ) or no HS ( NHS ) in Col - 0 wild type at HSP22 . 0 ( A ) and HSP70 ( B ) . Seedlings were subjected to ACC or a control treatment ( NHS ) 4 d after germination . At the indicated time points after the treatments , ChIP - qPCR was performed with antibodies against H3K9ac , H3K4me3 , H3K4me2 and H3 . Schematics show positions of regions analyzed . Amplicon positions relative to TSS are HSP22 . 0 : 1 , - 2570 bp ; 2 , + 235 bp . HSP70 : 1 , 4192 bp downstream of the 3 ' UTR ; 2 , + 47 bp . Data shown are averages over three biological replicates . Amplification values were normalized to input , H3 and 4 h NHS region 2 . The bottom panel shows the H3 signal normalized to input and 4 h NHS region 2 . Squares and triangles within bars mark significant differences ( p < 0 . 01 and p < 0 . 05 , respectively , Student ´ s t - test ) between ACC and NHS samples of the same time point . Error bars indicate SE .

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Dynamics of histone modifications after an acclimatizing HS (ACC) or no HS (NHS) in Col-0 wild type at HSP22.0 (A) and HSP70 (B). Seedlings were subjected to ACC or a control treatment (NHS) 4 d after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac, H3K4me3, H3K4me2 and H3. Schematics show positions of regions analyzed. Amplicon positions relative to TSS are HSP22.0: 1, -2570 bp; 2, +235 bp. HSP70: 1, 4192 bp downstream of the 3'UTR; 2, +47 bp. Data shown are averages over three biological replicates. Amplification values were normalized to input, H3 and 4 h NHS region 2. The bottom panel shows the H3 signal normalized to input and 4 h NHS region 2. Squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively, Student´s t-test) between ACC and NHS samples of the same time point. Error bars indicate SE.


A . Transcript levels of the indicated HS - inducible genes after ACC in Col - 0 ( blue bars ) and hsfa2 ( orange bars ) as determined by quantitative Reverse Transcription - PCR ( qRT - PCR ) . Transcript levels were normalized to TUB6 and the respective NHS harvested at the same time point ( [ GENE OF INTERESTACC x h / TUB6ACC x h ] / [ GENE OF INTERESTNHS x h / TUB6NHS x h ] ) . Note that the y - axis is on a log10 scale . Error bars show SE of at least three biological replicates . Asterisks show significant differences between the genotypes at the same time point based on Student ´ s t - test ( * : p < 0 . 05 ; * * : p < 0 . 01 ) .

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A. Transcript levels of the indicated HS-inducible genes after ACC in Col-0 (blue bars) and hsfa2 (orange bars) as determined by quantitative Reverse Transcription-PCR (qRT-PCR). Transcript levels were normalized to TUB6 and the respective NHS harvested at the same time point ([GENE OF INTERESTACC x h/ TUB6ACC x h]/ [GENE OF INTERESTNHS x h/ TUB6NHS x h]). Note that the y-axis is on a log10 scale. Error bars show SE of at least three biological replicates. Asterisks show significant differences between the genotypes at the same time point based on Student´s t-test (*: p<0.05; **: p<0.01).


B . Immunoblotting of HSP21 and HSP101 in Col - 0 ( C ) and hsfa2 ( h ) at the indicated time points after ACC or NHS . TUBULIN was used as a loading control . M , marker .

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B. Immunoblotting of HSP21 and HSP101 in Col-0 (C) and hsfa2 (h) at the indicated time points after ACC or NHS. TUBULIN was used as a loading control. M, marker.


H3K4me3 ( A ) or H3K4me2 ( B ) levels after an acclimatizing HS ( ACC ) in Col - 0 and hsfa2 at HSP18 . 2 , HSP22 . 0 , HSP70 and APX2 as detected by ChIP - qPCR . Col - 0 ( blue bars ) and hsfa2 ( orange bars ) seedlings were subjected to ACC or no treatment ( NHS ) 4 d after germination . At the indicated time points after the treatment , ChIP - qPCR was performed with antibodies against H3K4me3 ( A ) , H3K4me2 ( B ) and H3 ( for normalization ) . Schematics show positions of regions analyzed ( grey bars , UTR ; black bar , exons ) . Intergenic control region 1 is 3123 bp ( APX2 ) or 2570 bp ( HSP22 . 0 ) upstream of the TSS , or 5311 bp ( HSP18 . 2 ) or 6725 bp ( HSP70 ) downstream of the TSS , respectively . Data are averages over four biological replicates . Amplification values were normalized to input and H3 and the Col - 0 4h NHS region 2 ( HSP18 . 2 , HSP22 . 0 and HSP70 ) or region 3 ( for APX2 ) . * , p < 0 . 05 ; * * , p < 0 . 01 for differences between genotypes at the same time point and treatment ; squares and triangles within bars mark significant differences ( p < 0 . 01 and p < 0 . 05 , respectively ) between ACC and NHS samples of the same time point and genotype , Student ´ s t - test . Error bars indicate SE .

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

H3K4me3 (A) or H3K4me2 (B) levels after an acclimatizing HS (ACC) in Col-0 and hsfa2 at HSP18.2, HSP22.0, HSP70 and APX2 as detected by ChIP-qPCR. Col-0 (blue bars) and hsfa2 (orange bars) seedlings were subjected to ACC or no treatment (NHS) 4 d after germination. At the indicated time points after the treatment, ChIP-qPCR was performed with antibodies against H3K4me3 (A), H3K4me2 (B) and H3 (for normalization). Schematics show positions of regions analyzed (grey bars, UTR; black bar, exons). Intergenic control region 1 is 3123 bp (APX2) or 2570 bp (HSP22.0) upstream of the TSS, or 5311 bp (HSP18.2) or 6725 bp (HSP70) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4h NHS region 2 (HSP18.2, HSP22.0 and HSP70) or region 3 (for APX2). *, p<0.05; **, p<0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student´s t-test. Error bars indicate SE.


Figure 4 : Histone H3K9ac profiles after ACC in Col - 0 and hsfa2 at HSP18 . 2 , HSP22 . 0 , HSP70 and APX2 as detected by ChIP - qPCR . Col - 0 ( blue bars ) and hsfa2 ( orange bars ) seedlings were subjected to ACC or control treatment ( NHS ) 4 d after germination . At the indicated time points after the treatments , ChIP - qPCR was performed with antibodies against H3K9ac or H3 . Schematics ( grey bars , UTR ; black bar , exons ) show positions of regions analyzed . Intergenic control region 1 is 3123 bp ( APX2 ) or 2570 bp ( HSP22 . 0 ) upstream of the TSS , or 5311 bp ( HSP18 . 2 ) or 6725 bp ( HSP70 ) downstream of the TSS , respectively . Data are averages over four biological replicates . Amplification values were normalized to input and H3 and the Col - 0 4h NHS region 2 ( HSP18 . 2 , HSP22 . 0and HSP70 ) or 3 ( for APX2 ) . * , p < 0 . 05 ; * * , p < 0 . 01 for differences between genotypes at the same time point and treatment ; squares and triangles within bars mark significant differences ( p < 0 . 01 and p < 0 . 05 , respectively ) between ACC and NHS samples of the same time point and genotype , Student ´ s t - test . Error bars indicate SE .

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

Figure 4: Histone H3K9ac profiles after ACC in Col-0 and hsfa2 at HSP18.2, HSP22.0, HSP70 and APX2 as detected by ChIP-qPCR.Col-0 (blue bars) and hsfa2 (orange bars) seedlings were subjected to ACC or control treatment (NHS) 4 d after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac or H3. Schematics (grey bars, UTR; black bar, exons) show positions of regions analyzed. Intergenic control region 1 is 3123 bp (APX2) or 2570 bp (HSP22.0) upstream of the TSS, or 5311 bp (HSP18.2) or 6725 bp (HSP70) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4h NHS region 2 (HSP18.2, HSP22.0and HSP70) or 3 (for APX2). *, p<0.05; **, p<0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student´s t-test. Error bars indicate SE.