Figure 7 ( 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 . ( 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 ) . ( 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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Figure 7(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.(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).(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.


Figure 8 ( 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 . ( 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 . ( 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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Figure 8(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.(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.(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.


Figure 9 ( 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 . ( 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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Figure 9(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.(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.


Figure 8A . 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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Figure 8A. 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


Figure 9B . 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 . 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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Figure 9B. 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.D. Expression of Vps35 variants in cells from B was analyzed by SDS-PAGE and Western blot against Vps35. Tubulin served as loading control.


Figure 3A 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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Figure 3A 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.


Figure 4A 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 . 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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Figure 4A 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.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.


Figure 6A 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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Figure 6A 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.


Figure 1A . 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 . 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 . 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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Figure 1A. 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.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.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.


Figure 2A . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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.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.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.


Figure 3A . 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 . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. 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.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.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.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.


Figure 4A . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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.


Figure 5A . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. 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.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.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.


Figure 6A . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. 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).


Figure 7A . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. 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).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.


Figure 8A . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. 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.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.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.


Figure 1Dynamics 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Dynamics 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.


Figure 2A . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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.


Figure 3H3K4me3 ( 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3H3K4me3 (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 4Figure 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Figure 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.


Figure 5Figure 5 : HSFA2 associates with HS memory - related genes transiently after an acclimatizing HS . Binding of HSFA2 - YFP to the promoter regions of HSP18 . 2 ( A ) , HSP22 . 0 ( B ) , APX2 ( C ) , HSP70 ( D ) and ACTIN7 ( E ) was determined by ChIP - qPCR . Quantifications represent the enrichment relative to input in % . Red - orange - yellow bars represent time points ( 0 . 5 h - 4 h - 28 h ) after a ACC and grey bars represent the respective NHS control samples . Schematics show the promoter region of the respective gene with regions analyzed ( arrow , TSS ; grey bar , 5 ' UTR ; black bar , exon ; black boxes , heat shock elements ( 5 ' - nnGnAnnTnCtn - 3 ' ) . Regions are drawn to scale . Intergenic control region 1 is 4610 bp ( HSP18 . 2 ) and 4192 bp ( HSP70 ) downstream of the 3 ' UTR and 2570 bp ( HSP22 . 0 ) or 3123 bp ( APX2 ) upstream of the TSS , respectively . One representative experiment out of at least four biological replicates is shown . Error bars are SD of three replicates .

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Figure 5Figure 5: HSFA2 associates with HS memory-related genes transiently after an acclimatizing HS.Binding of HSFA2-YFP to the promoter regions of HSP18.2 (A), HSP22.0 (B), APX2 (C), HSP70 (D) and ACTIN7 (E) was determined by ChIP-qPCR. Quantifications represent the enrichment relative to input in %. Red-orange-yellow bars represent time points (0.5 h-4 h-28 h) after a ACC and grey bars represent the respective NHS control samples. Schematics show the promoter region of the respective gene with regions analyzed (arrow, TSS; grey bar, 5'UTR; black bar, exon; black boxes, heat shock elements (5'-nnGnAnnTnCtn-3'). Regions are drawn to scale. Intergenic control region 1 is 4610 bp (HSP18.2) and 4192 bp (HSP70) downstream of the 3'UTR and 2570 bp (HSP22.0) or 3123 bp (APX2) upstream of the TSS, respectively. One representative experiment out of at least four biological replicates is shown. Error bars are SD of three replicates.


Figure 6Figure 6 : Expression profiles of HS memory - related genes after a recurring HS show HSFA2 - dependent transcriptional memoryResponse of selected HS - inducible loci upon recurring HS as determined by qRT - PCR . Transcript levels of APX2 ( A ) , HSP18 . 2 ( B ) , HSP22 . 0 ( C ) , and HSP70 ( D ) in Col - 0 ( blue bars ) and hsfa2 ( orange bars ) at the indicated time points after a single HS or two HS separated by 2 d . Plants were subjected to 37 ° C for 60 min on either d 4 , d 6 or on d 4 + d 6 after germination . NHS samples were harvested at the same time points as HS d 4 samples . Transcript levels of four biological replicates normalized to TUB6 are shown . Small brackets compare the two genotypes at the same condition , large brackets compare the same genotype across two treatments ( at 1 h after treatment ) with Col - 0 indicated first ( Col - 0 / hsfa2 ) . Error bars show SE over four biological replicates . * , p < 0 . 05 ; * * , p < 0 . 01 ; Student ´ s t - test .

Figure 6Figure 6: Expression profiles of HS memory-related genes after a recurring HS show HSFA2-dependent transcriptional memoryResponse of selected HS-inducible loci upon recurring HS as determined by qRT-PCR. Transcript levels of APX2 (A), HSP18.2 (B), HSP22.0 (C), and HSP70 (D) in Col-0 (blue bars) and hsfa2 (orange bars) at the indicated time points after a single HS or two HS separated by 2 d. Plants were subjected to 37°C for 60 min on either d 4, d 6 or on d 4 + d 6 after germination. NHS samples were harvested at the same time points as HS d 4 samples. Transcript levels of four biological replicates normalized to TUB6 are shown. Small brackets compare the two genotypes at the same condition, large brackets compare the same genotype across two treatments (at 1 h after treatment) with Col-0 indicated first (Col-0/ hsfa2). Error bars show SE over four biological replicates. *, p<0.05; **, p<0.01; Student´s t-test.


Figure 1b ) Histograms of GFP expression ( x - axis ) are presented from A549 cells transduced with the different viral vectors . c ) Percentage of GFP positive cells from the experiment described in ( b ) . . d ) A PCR assay along with primers , as illustrated in Appendix Figure S2a , was used to detect formation of LTR junction and thereby circularization of the vector . The expected size of PCR product is 552 bp from a 1 - LTR circle and 1029 bp from a 2 - LTR circle . . e ) Southern Blot analysis to detect integration events in LV - and NILV - S / MAR - transduced single cell clones with DNA probed against the viral genome . A 6 . 8 kb positive control from a linearized plasmid ( lane 3 ) was included for indication of the size of DNA circles formed by non - integrated NILV - S / MAR vector . f - h ) A padlock probe assay , as illustrated in Appendix Figure S4 , was used to demonstrate persistence of proviral DNA either in a circular or integrated form . Representative images show DNA persistence in ( f ) LV - , ( g ) NILV - or ( h ) NILV - S / MAR - transduced cells . Each red dot ( Cy3 ) represents a single DNA molecule and cell nuclei were stained with Hoechst 33342 .

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Figure 1b) Histograms of GFP expression (x-axis) are presented from A549 cells transduced with the different viral vectors.c) Percentage of GFP positive cells from the experiment described in (b).. d) A PCR assay along with primers, as illustrated in Appendix Figure S2a, was used to detect formation of LTR junction and thereby circularization of the vector. The expected size of PCR product is 552 bp from a 1-LTR circle and 1029 bp from a 2-LTR circle.. e) Southern Blot analysis to detect integration events in LV- and NILV-S/MAR-transduced single cell clones with DNA probed against the viral genome. A 6.8 kb positive control from a linearized plasmid (lane 3) was included for indication of the size of DNA circles formed by non-integrated NILV-S/MAR vector.f-h) A padlock probe assay, as illustrated in Appendix Figure S4, was used to demonstrate persistence of proviral DNA either in a circular or integrated form. Representative images show DNA persistence in (f) LV-, (g) NILV- or (h) NILV-S/MAR-transduced cells. Each red dot (Cy3) represents a single DNA molecule and cell nuclei were stained with Hoechst 33342.


Figure 2c ) Flow cytometry histograms of RFP expression ( x - axis ) of A549 / RFP + Luc + cells at day 20 and 27 after NILV - S / MAR ( GFP , shNT ) or NILV - S / MAR ( GFP , shRFP ) transduction . d ) Luciferase activity of the A549 / RFP + Luc + cells at day 20 and day 44 after NILV - S / MAR ( GFP , shNT ) - or NILV - S / MAR ( GFP , shRFP ) - transduction . e - h ) Representative fluorescence microscopy images show the expression of RFP from either NILV - S / MAR - transduced or un - transduced A549 / RFP + Luc + cells with cell nuclei stained with Hoechst 33342 ( blue ) . RFP signal ( orange ) were collected with a fixed exposure time ( 300 ms ) for equal comparison .

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Figure 2c) Flow cytometry histograms of RFP expression (x-axis) of A549/RFP+Luc+ cells at day 20 and 27 after NILV-S/MAR(GFP, shNT) or NILV-S/MAR(GFP, shRFP) transduction.d) Luciferase activity of the A549/RFP+Luc+ cells at day 20 and day 44 after NILV-S/MAR(GFP, shNT)- or NILV-S/MAR(GFP, shRFP)-transduction.e-h) Representative fluorescence microscopy images show the expression of RFP from either NILV-S/MAR-transduced or un-transduced A549/RFP+Luc+ cells with cell nuclei stained with Hoechst 33342 (blue). RFP signal (orange) were collected with a fixed exposure time (300 ms) for equal comparison.


Figure 3c ) Percentage of CD19CAR expression on CD3 + T cells from each independent donor . d ) Histograms of CD19 CAR expression on T cells form one representative donor before and after expansion . e ) CD107a expression on transduced T cells ( CD3 + ) after exposure to CD19 + target cells ( Daudi ) for 24 h ( n = 6 ) . f ) IFN - γ production in the supernatant was analyzed by ELISA after 24 h of exposure of transduced T cells to target cells ( n = 6 ) . g ) Relative viability of target cells ( CD19 + Daudi ) , as an indication of T cell killing efficacy , were measured at different effector T cell to target cell ratio ( E : T ratio ) and normalized to the signal from target cells alone ( n = 6 ) . h ) Transduced T cells were labeled with a fluorescent cell membrane dye and co - cultured with CD19 + Daudi cells for 4 days . Dye dilution upon T cell division was monitored by flow cytometry and representative histograms are shown . i - m ) Mice were treated by intravenous injection of NILV - S / MAR ( Mock ) , LV ( CD19CAR ) and NILV - S / MAR ( CD19CAR ) - transduced T cells ( 8x106 cells / injection ) at days 18 , 21 , 24 after tumor implantation . Tumor growth was monitored by caliper measurements . i - k ) Tumor volume of individual mice in each treatment groups are presented . l ) The average tumor volume of each treatment group is presented and the values on day 37 were statistically compared ( n = 12 in LV group , n = 9 in NILV - S / MAR group and n = 11 in Mock control group ) . m ) The survival of mice is shown as Kaplan - Meier curves . n - p ) Representative images showing infiltration of adoptively transferred human T cells into tumors . T cells were visualized by staining for human CD3 ( red ) and nucleus ( blue ) . q ) Quantification of CD3 + T cells from each treatment group is presented ( n = 5 ) .

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Figure 3c) Percentage of CD19CAR expression on CD3+ T cells from each independent donor. d) Histograms of CD19 CAR expression on T cells form one representative donor before and after expansion. e) CD107a expression on transduced T cells (CD3+) after exposure to CD19+ target cells (Daudi) for 24 h (n=6). f) IFN-γ production in the supernatant was analyzed by ELISA after 24 h of exposure of transduced T cells to target cells (n=6). g) Relative viability of target cells (CD19+ Daudi), as an indication of T cell killing efficacy, were measured at different effector T cell to target cell ratio (E:T ratio) and normalized to the signal from target cells alone (n=6). h) Transduced T cells were labeled with a fluorescent cell membrane dye and co-cultured with CD19+ Daudi cells for 4 days. Dye dilution upon T cell division was monitored by flow cytometry and representative histograms are shown.i-m) Mice were treated by intravenous injection of NILV-S/MAR(Mock), LV(CD19CAR) and NILV-S/MAR(CD19CAR)-transduced T cells (8x106 cells/injection) at days 18, 21, 24 after tumor implantation. Tumor growth was monitored by caliper measurements. i-k) Tumor volume of individual mice in each treatment groups are presented. l) The average tumor volume of each treatment group is presented and the values on day 37 were statistically compared (n=12 in LV group, n=9 in NILV-S/MAR group and n=11 in Mock control group). m) The survival of mice is shown as Kaplan-Meier curves.n-p) Representative images showing infiltration of adoptively transferred human T cells into tumors. T cells were visualized by staining for human CD3 (red) and nucleus (blue). q) Quantification of CD3+ T cells from each treatment group is presented (n=5).


Figure 1Percent body weight of male Prdx4 WT and KO mice over the 72 h course of LPS ( 4 . 5 mg / kg BW ) or PBS injection ( i . p . ) . Each circle represents a mean of n = 7 mice , vertical lines indicate SEMCytokine concentration in Prdx4 WT and KO mice in response to LPS injection . ( B ) Cxcl1 levels in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . ( C ) TNF - α levels in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . ( D ) IL - 1β levels in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . Each dot ( B - D ) represents an individual mouse . Horizontal lines indicate mean .

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Figure 1Percent body weight of male Prdx4 WT and KO mice over the 72 h course of LPS (4.5 mg/kg BW) or PBS injection (i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEMCytokine concentration in Prdx4 WT and KO mice in response to LPS injection. (B) Cxcl1 levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (C) TNF-α levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (D) IL-1β levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. Each dot (B-D) represents an individual mouse. Horizontal lines indicate mean.


Figure 2Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS ( 4 . 5 mg / kg BW ) injection ( i . p . ) and treatment with IL - 1 receptor antagonist ( IL - 1RA ) Anakinra ( 200 µg / mouse ) or control . Arrows indicate time point of Anakinra injection . Each circle represents a mean of n = 5 mice , vertical lines indicate SEM . Serum concentration of Cxcl1 , TNF - α and IL - 1β in Prdx4 WT and KO mice injected with LPS , LPS and IL - 1RA or control . Horizontal lines indicate mean . Percent body weight of male Prdx4 - flox and Prdx4 - ∆ LysMCre mice over the 48 h course of 4 . 5 mg / kg BW LPS ( i . p . ) . Each circle represents a mean of n = 7 mice , vertical lines indicate SEM . Serum concentration of Cxcl1 , TNF - α and IL - 1β in Prdx4 - flox and Prdx4 - ∆ LysMCre mice injected with LPS . Each dot ( B , D ) represents an individual mouse . Horizontal lines indicate mean .

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Figure 2Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS (4.5 mg/kg BW) injection (i.p.) and treatment with IL-1 receptor antagonist (IL-1RA) Anakinra (200 µg/mouse) or control. Arrows indicate time point of Anakinra injection. Each circle represents a mean of n=5 mice, vertical lines indicate SEM.Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4 WT and KO mice injected with LPS, LPS and IL-1RA or control. Horizontal lines indicate mean.Percent body weight of male Prdx4-flox and Prdx4-∆LysMCre mice over the 48 h course of 4.5 mg/kg BW LPS ( i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM.Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4-flox and Prdx4-∆LysMCre mice injected with LPS. Each dot (B, D) represents an individual mouse. Horizontal lines indicate mean.


Figure 3Concentration of Cxcl1 , TNF - α and IL - 1β in the supernatants of Prdx4 WT and KO BMDMs in response to a time course of LPS stimulation ( 100 ng / ml LPS , time points indicated ) . IL - 1β release of Prdx4 WT and KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) and then pulsed for indicated time points with ATP ( 5 mM ) . Western blot analysis of IL - 1β in cell lysates and supernatants of Prdx4 WT and KO BMDMs , primed with LPS ( 100 ng / ml ) and pulsed with ATP ( 5 mM ) for 4 h or left untreated . Dashed line indicates vertical slice . Immunofluorescence microscopy of ASC speck formation in Prdx4 WT and KO BMDMs in response to Nigericin ( 10 µg / ml ) stimulation for 45 min of LPS - primed cells . Cells were stained with an antibody to ASC and nuclei were counterstained using DAPI . Scale bar indicates 20 µm . ASC speck - positive cells were counted and expressed as percentage of total cells . Bars represent a mean of n = 4 mice , vertical lines indicate SD . n . s . not significant ( two - tailed t - test ) . Western blot analysis of caspase - 1 cleavage in the supernatant of Prdx4 WT and KO BMDMs in response to Nigericin ( 10 µg / ml ) stimulation for 1 h after priming with LPS ( 100 ng / ml ) , LPS - priming alone or without stimulation . Whole cell lysates were analyzed for pro - caspase - 1 and Gapdh levels . IL - 1β release in Prdx4 WT and KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) and then pulsed for 3 h with ATP ( 5 mM ) or Nigericin ( 10 µg / ml ) or transfected for 3 h with poly ( dA : dT ) or Flagellin ( 1 µg / ml each ) or treated with transfection agent only . Quantification of cell death by LDH - release in Prdx4 WT and KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) and then pulsed for 3 h with ATP ( 5 mM ) or Nigericin ( 10 µg / ml ) or transfected for 3 h with poly ( dA : dT ) or flagellin ( 1 µg / ml each ) or treated with transfection agent only .

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Figure 3Concentration of Cxcl1, TNF-α and IL-1β in the supernatants of Prdx4 WT and KO BMDMs in response to a time course of LPS stimulation (100 ng/ml LPS, time points indicated).IL-1β release of Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for indicated time points with ATP (5 mM).Western blot analysis of IL-1β in cell lysates and supernatants of Prdx4 WT and KO BMDMs, primed with LPS (100 ng/ml) and pulsed with ATP (5 mM) for 4 h or left untreated. Dashed line indicates vertical slice.Immunofluorescence microscopy of ASC speck formation in Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 45 min of LPS-primed cells. Cells were stained with an antibody to ASC and nuclei were counterstained using DAPI. Scale bar indicates 20 µm. ASC speck-positive cells were counted and expressed as percentage of total cells. Bars represent a mean of n=4 mice, vertical lines indicate SD. n.s. not significant (two-tailed t-test).Western blot analysis of caspase-1 cleavage in the supernatant of Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 1 h after priming with LPS (100 ng/ml), LPS-priming alone or without stimulation. Whole cell lysates were analyzed for pro-caspase-1 and Gapdh levels.IL-1β release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or Flagellin (1 µg/ml each) or treated with transfection agent only.Quantification of cell death by LDH-release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or flagellin (1 µg/ml each) or treated with transfection agent only.


Figure 4Western blot analysis of rPRDX4 , rCASP - 1 and co - incubated rPRDX4 and rCASP - 1 after non - reducing SDS - PAGE . Black arrows at 250 kDa indicate the corresponding molecular weights of rCASP - 1 and rPRDX4 oligomers and decrease of the Prdx4 band intensity upon co - incubation with rCASP - 1 . Black arrow at approx . 50 kDa indicates the appearance of an additional rPRDX4 band which corresponds to the molecular weight of a Prdx4 dimer . Western blot analysis of rPRDX4 ( = P ) and rCASP - 1 ( = C ) after alkylation ( = A ) and non - reducing SDS - PAGE . Black arrows indicate the termination of the integration of rCASP - 1 into the rPRDX4 decamer / HMWC upon alkylation . Rate of caspase - 1 activity in the presence of non - reduced decameric rPrdx4 , reduced dimeric and monomeric rPRDX4 , YVAD or control . Foldchange in IL - 1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3 , ASC , IL - 1β and caspase - 1 WT or Cys - to - Ser mutants C362S or C397S and co - transfected with Prdx4 or control . Cells were stimulated with 2 . 5 mM ATP for 30 min before analysis . Western blot analysis of non - reducing SDS - PAGE of cell lysates from HEK293 cells transfected with caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants and co - transfected with Prdx4 - GFP or GFP as control . Western blot analysis of co - immunoprecipitation using HA - magnetic beads from cell lysates of HEK293 cells transfected with HA - tagged caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants and co - transfected with Prdx4 - GFP or GFP as control . IL - 1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3 , ASC , IL - 1β and caspase - 1 and co - transfected with Prdx4 WT or Cys - to - Ala mutants C51A , C124A , C245A or DM C124A / C245A or control . Cells were stimulated with 2 . 5 mM ATP for 30 min before analysis . Western blot analysis of co - immunoprecipitation using HA - magnetic beads from cell lysates of HEK293 cells transfected with HA - tagged caspase - 1 WT or control and co - transfected with Prdx4 WT or Cys - to - Ala mutants C51A , C124A , C245A or DM C124A / C245A .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Western blot analysis of rPRDX4, rCASP-1 and co-incubated rPRDX4 and rCASP-1 after non-reducing SDS-PAGE. Black arrows at 250 kDa indicate the corresponding molecular weights of rCASP-1 and rPRDX4 oligomers and decrease of the Prdx4 band intensity upon co-incubation with rCASP-1. Black arrow at approx. 50 kDa indicates the appearance of an additional rPRDX4 band which corresponds to the molecular weight of a Prdx4 dimer.Western blot analysis of rPRDX4 (=P) and rCASP-1 (=C) after alkylation (=A) and non-reducing SDS-PAGE. Black arrows indicate the termination of the integration of rCASP-1 into the rPRDX4 decamer/HMWC upon alkylation.Rate of caspase-1 activity in the presence of non-reduced decameric rPrdx4, reduced dimeric and monomeric rPRDX4, YVAD or control.Foldchange in IL-1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3, ASC, IL-1β and caspase-1 WT or Cys-to-Ser mutants C362S or C397S and co-transfected with Prdx4 or control. Cells were stimulated with 2.5 mM ATP for 30 min before analysis.Western blot analysis of non-reducing SDS-PAGE of cell lysates from HEK293 cells transfected with caspase-1 WT, C362S, C397S, or C362S plus C397S mutants and co-transfected with Prdx4-GFP or GFP as control.Western blot analysis of co-immunoprecipitation using HA-magnetic beads from cell lysates of HEK293 cells transfected with HA-tagged caspase-1 WT, C362S, C397S, or C362S plus C397S mutants and co-transfected with Prdx4-GFP or GFP as control.IL-1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3, ASC, IL-1β and caspase-1 and co-transfected with Prdx4 WT or Cys-to-Ala mutants C51A, C124A, C245A or DM C124A/C245A or control. Cells were stimulated with 2.5 mM ATP for 30 min before analysis.Western blot analysis of co-immunoprecipitation using HA-magnetic beads from cell lysates of HEK293 cells transfected with HA-tagged caspase-1 WT or control and co-transfected with Prdx4 WT or Cys-to-Ala mutants C51A, C124A, C245A or DM C124A/C245A.


Figure 5Western blot analysis of Prdx4 , pro - caspase ‑ 1 , Gapdh and E - Cadherin from the cytosolic and insoluble cell fraction of LPS and / or ATP - stimulated BMDMs or untreated controls . Prdx4 concentration in supernatants of Prdx4 WT or KO BMDMs , primed for 6 h with LPS and pulsed for indicated time points with 5 mM ATP . Each circle represents a mean of n = 3 mice , vertical lines indicate SD . * * , p < 0 . 01 ; n . s . not significant ( two - tailed t - test ) . Concentration of Prdx4 in the serum of WT mice , injected with LPS ( 4 . 5 mg / kg BW ) for the time points indicated . Each dot represents an individual mouse . Horizontal lines indicate mean . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed t - test ) . Concentration of Prdx4 in supernatants of Prdx4 WT and KO BMDMs . Cells were primed with LPS ( 100 ng / ml ) for 6 h , followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min and stimulated with 5 mM ATP for 4 h or no further stimulation . Each bar represents a mean of n = 3 mice , vertical lines indicate SD . * * , p < 0 . 01 ; n . s . not significant ( two - tailed t - test ) . Relative levels of Prdx4 secretion in response to LPS + ATP stimulation and pretreatment with either Glycine , NSA or GW4869 and relative levels of caspase - 1 secretion in response to LPS + ATP stimulation and pretreatment with in response to LPS + ATP stimulation and pretreatment with GW4869 . Each bar represents a mean of n = 3 biological with 2 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . not significant ( two - tailed t - test ) . Western blot analysis of subcellular organelle fractions . OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS ( 100 ng / ml ) for 12 h and stimulated with 5 mM ATP for 4 h .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Western blot analysis of Prdx4, pro-caspase‑1, Gapdh and E-Cadherin from the cytosolic and insoluble cell fraction of LPS and/or ATP-stimulated BMDMs or untreated controls.Prdx4 concentration in supernatants of Prdx4 WT or KO BMDMs, primed for 6 h with LPS and pulsed for indicated time points with 5 mM ATP. Each circle represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test).Concentration of Prdx4 in the serum of WT mice, injected with LPS (4.5 mg/kg BW) for the time points indicated. Each dot represents an individual mouse. Horizontal lines indicate mean. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).Concentration of Prdx4 in supernatants of Prdx4 WT and KO BMDMs. Cells were primed with LPS (100 ng/ml) for 6 h, followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min and stimulated with 5 mM ATP for 4 h or no further stimulation. Each bar represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test).Relative levels of Prdx4 secretion in response to LPS+ATP stimulation and pretreatment with either Glycine, NSA or GW4869 and relative levels of caspase-1 secretion in response to LPS+ATP stimulation and pretreatment with in response to LPS+ATP stimulation and pretreatment with GW4869. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; n.s. not significant (two-tailed t-test).Western blot analysis of subcellular organelle fractions. OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS (100 ng/ml) for 12 h and stimulated with 5 mM ATP for 4 h.