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

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

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

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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 p ‐ eIF2α in Mfn2 KO MEFs , Mfn2 knockdown 3T3 ‐ L1 fibroblasts , 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 of p ‐ PERK p ‐ eIF2α in MEFs . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT .

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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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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 of Mbp positive Schwann cellnuclei , n = 9 ( NT ) , n = 8 ( 1mM ) , n = 9 ( 5mM ) , n = 8 ( 10mM ) , DRGs / coverslips . Representative of three independent experiments , p = 0 . 0055 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc .

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

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

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


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

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

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

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


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

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

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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


C . Representative of semithin section of WT Pmp22 - / - sciatic nerves at P30 . Tomacula are abundant in Pmp22 - / - nerves at this , where both with thicker myelin ( in range of < 3 - 4 μm in diameter ) thinner myelin ( with diameters greater than 4 μm , particularly in motor fasicles ) can be observed . C ' . Niaspan does not ameliorate phenotype in Pmp22 - / - mice , as indicated by number of tomacula ( of of tomacula in entire sciatic nerve section , results are mean ± , p = 0 . 7400 , Mann Whitney U , n = 15 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 starting at P15 for 15 days . A significant time by group effect is noted between WT saline Pmp22 - / - saline ( group effect p = 0 . 9907 time effect p = 0 . 0057 ) , indicating that two groups start with similar values but then WT saline group ( n = 6 ) grows more in time . When Pmp22 - / - saline ( n = 5 ) Pmp22 - / - Niaspan - treated ( n = 6 ) are compared , group effect is statistically significant ( p = 0 . 0076 ) while 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 .

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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 - , with axonal diameter distribution : WT 0 . 68 ± 0 . 003 , 2945 , n = 5 ; Pmp22 - / - 0 . 64 ± 0 . 003 , 1557 , n = 6 ; Pmp22 - / - Niaspan 0 . 63 ± 0 . 003 , 1445 , n = 6 , results are mean ± . 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 loss of myelinated in Pmp22 - / - nerves for 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 ) HSP70 ( B ) . Seedlings were subjected to ACC or a control treatment ( NHS ) 4 d after germination . At indicated time points after treatments , ChIP - qPCR was performed with antibodies against H3K9ac , H3K4me3 , H3K4me2 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 3 ' UTR ; 2 , + 47 bp . Data shown are averages over three biological replicates . Amplification values were normalized to input , H3 4 h NHS region 2 . bottom panel shows H3 signal normalized to input 4 h NHS region 2 . Squares triangles within bars mark significant differences ( p < 0 . 01 p < 0 . 05 , respectively , Student ´ s t - ) between ACC NHS samples of 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 of indicated HS - inducible genes after ACC in Col - 0 ( blue bars ) hsfa2 ( orange bars ) as determined by quantitative Reverse Transcription - PCR ( qRT - PCR ) . Transcript were normalized to TUB6 respective NHS harvested at same time point ( [ GENE OF INTERESTACC x h / TUB6ACC x h ] / [ GENE OF INTERESTNHS x h / TUB6NHS x h ] ) . Note that y - axis is on a log10 scale . Error bars show SE of at least three biological replicates . Asterisks show significant differences between genotypes at same time point on Student ´ s t - ( * : 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 HSP101 in Col - 0 ( C ) hsfa2 ( h ) at 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 ) after an acclimatizing HS ( ACC ) in Col - 0 hsfa2 at HSP18 . 2 , HSP22 . 0 , HSP70 APX2 as detected by ChIP - qPCR . Col - 0 ( blue bars ) hsfa2 ( orange bars ) seedlings were subjected to ACC or no treatment ( NHS ) 4 d after germination . At indicated time points after treatment , ChIP - qPCR was performed with antibodies against H3K4me3 ( A ) , H3K4me2 ( B ) 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 TSS , or 5311 bp ( HSP18 . 2 ) or 6725 bp ( HSP70 ) downstream of TSS , respectively . Data are averages over four biological replicates . Amplification values were normalized to input H3 Col - 0 4h NHS region 2 ( HSP18 . 2 , HSP22 . 0 HSP70 ) or region 3 ( for APX2 ) . * , p < 0 . 05 ; * * , p < 0 . 01 for differences between genotypes at same time point treatment ; squares triangles within bars mark significant differences ( p < 0 . 01 p < 0 . 05 , respectively ) between ACC NHS samples of same time point genotype , Student ´ s t - . Error bars indicate SE .

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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 hsfa2 at HSP18 . 2 , HSP22 . 0 , HSP70 APX2 as detected by ChIP - qPCR . Col - 0 ( blue bars ) hsfa2 ( orange bars ) seedlings were subjected to ACC or control treatment ( NHS ) 4 d after germination . At indicated time points after 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 TSS , or 5311 bp ( HSP18 . 2 ) or 6725 bp ( HSP70 ) downstream of TSS , respectively . Data are averages over four biological replicates . Amplification values were normalized to input H3 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 same time point treatment ; squares triangles within bars mark significant differences ( p < 0 . 01 p < 0 . 05 , respectively ) between ACC NHS samples of same time point genotype , Student ´ s t - . Error bars indicate SE .

O O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O 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-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


Figure 5 : HSFA2 associates with HS memory - related genes transiently after an acclimatizing HS . Binding of HSFA2 - YFP to promoter regions of HSP18 . 2 ( A ) , HSP22 . 0 ( B ) , APX2 ( C ) , HSP70 ( D ) ACTIN7 ( E ) was determined by ChIP - qPCR . Quantifications represent enrichment relative to input in % . Red - orange - yellow bars represent time points ( 0 . 5 h - 4 h - 28 h ) after a ACC grey bars represent respective NHS control samples . Schematics show promoter region of 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 ) 4192 bp ( HSP70 ) downstream of 3 ' UTR 2570 bp ( HSP22 . 0 ) or 3123 bp ( APX2 ) upstream of 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 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 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 of APX2 ( A ) , HSP18 . 2 ( B ) , HSP22 . 0 ( C ) , HSP70 ( D ) in Col - 0 ( blue bars ) hsfa2 ( orange bars ) at indicated time points after a single HS or two HS separated by 2 d . 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 same time points as HS d 4 samples . Transcript of four biological replicates normalized to TUB6 are shown . Small brackets compare two genotypes at same condition , large brackets compare 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 - .

O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O 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-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O 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 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.


b ) Histograms of GFP expression ( x - axis ) are presented from A549 transduced with different viral vectors .

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b) Histograms of GFP expression (x-axis) are presented from A549 cells transduced with the different viral vectors.


c ) of GFP positive from experiment described in ( b ) .

c) Percentage of GFP positive cells from the experiment described in (b).


. d ) A PCR along with primers , as illustrated in Appendix Figure S2a , was used to detect formation of LTR junction thereby circularization of vector . expected of PCR product is 552 bp from a 1 - LTR circle 1029 bp from a 2 - LTR circle .

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. 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 to detect integration events in LV - NILV - S / MAR - transduced single clones with probed against viral genome . A 6 . 8 kb positive control from a linearized plasmid ( lane 3 ) was included for indication of of circles formed by non - integrated NILV - S / MAR vector .

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. 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 , as illustrated in Appendix Figure S4 , was used to demonstrate persistence of proviral either in a circular or integrated form . Representative show persistence in ( f ) LV - , ( g ) NILV - or ( h ) NILV - S / MAR - transduced . Each red dot ( Cy3 ) represents a single molecule nuclei were stained with Hoechst 33342 .

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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.


c ) Flow cytometry histograms of RFP expression ( x - axis ) of A549 / RFP + Luc + at day 20 27 after NILV - S / MAR ( GFP , shNT ) or NILV - S / MAR ( GFP , shRFP ) transduction .

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c) 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 A549 / RFP + Luc + at day 20 day 44 after NILV - S / MAR ( GFP , shNT ) - or NILV - S / MAR ( GFP , shRFP ) - transduction .

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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 microscopy show expression of RFP from either NILV - S / MAR - transduced or un - transduced A549 / RFP + Luc + with 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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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.


c ) of CD19CAR expression on CD3 + T from each independent donor . d ) Histograms of CD19 CAR expression on T form one representative donor before after expansion . e ) CD107a expression on transduced T ( CD3 + ) after exposure to CD19 + target ( Daudi ) for 24 h ( n = 6 ) . f ) IFN - γ production in supernatant was analyzed by ELISA after 24 h of exposure of transduced T to target ( n = 6 ) . g ) Relative viability of target ( CD19 + Daudi ) , as an indication of T killing efficacy , were measured at different effector T to target ( E : T ) normalized to signal from target alone ( n = 6 ) . h ) Transduced T were labeled with a dye co - cultured with CD19 + Daudi for 4 days . Dye dilution upon T division was monitored by flow cytometry representative histograms are shown .

O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

c) 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 ) NILV - S / MAR ( CD19CAR ) - transduced T ( 8x106 / injection ) at days 18 , 21 , 24 after implantation . growth was monitored by caliper measurements . i - k ) volume of mice in each treatment groups are presented . l ) average volume of each treatment group is presented values on day 37 were statistically compared ( n = 12 in LV group , n = 9 in NILV - S / MAR group n = 11 in Mock control group ) . m ) survival of mice is shown as Kaplan - Meier curves .

O O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O

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 showing infiltration of adoptively transferred human T into tumors . T were visualized by for human CD3 ( red ) nucleus ( blue ) . q ) of CD3 + T from each treatment group is presented ( n = 5 ) .

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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).


of Prdx4 WT KO mice over 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

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Percent 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 SEM


Cytokine in Prdx4 WT KO mice in response to LPS injection . ( B ) Cxcl1 in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . ( C ) TNF - α in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . ( D ) IL - 1β in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . Each dot ( B - D ) represents an mouse . Horizontal lines indicate mean .

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Cytokine 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.


of Prdx4 WT KO mice over 48 h course of LPS ( 4 . 5 mg / kg BW ) injection ( i . p . ) 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 .

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Percent 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 of Cxcl1 , TNF - α IL - 1β in Prdx4 WT KO mice injected with LPS , LPS IL - 1RA or control . Horizontal lines indicate mean .

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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.


of Prdx4 - flox Prdx4 - ∆ LysMCre mice over 48 h course of 4 . 5 mg / kg BW LPS ( i . p . ) . Each circle represents a mean of n = 7 mice , vertical lines indicate .

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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 of Cxcl1 , TNF - α IL - 1β in Prdx4 - flox Prdx4 - ∆ LysMCre mice injected with LPS . Each dot ( B , D ) represents an mouse . Horizontal lines indicate mean .

B-TISSUE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O O O

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.


of Cxcl1 , TNF - α IL - 1β in supernatants of Prdx4 WT KO BMDMs in response to a time course of LPS stimulation ( 100 ng / ml LPS , time points indicated ) .

O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O

Concentration 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 KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) then pulsed for indicated time points with ATP ( 5 mM ) .

B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

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 of IL - 1β in lysates supernatants of Prdx4 WT KO BMDMs , primed with LPS ( 100 ng / ml ) pulsed with ATP ( 5 mM ) for 4 h or left untreated . Dashed indicates vertical slice .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

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 KO BMDMs in response to Nigericin ( 10 µg / ml ) stimulation for 45 min of LPS - primed . were stained with an antibody to ASC nuclei were counterstained using DAPI . Scale bar indicates 20 µm . ASC speck - positive were counted expressed as of total . Bars represent a mean of n = 4 mice , vertical lines indicate SD . n . s . not significant ( two - t - ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O B-GENEPROD B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

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 of caspase - 1 cleavage in supernatant of Prdx4 WT 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 lysates were analyzed for pro - caspase - 1 Gapdh .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

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 KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) 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 .

B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O

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.


of by LDH - release in Prdx4 WT KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) 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 .

O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O

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.


Western blot of rPRDX4 , rCASP - 1 co - incubated rPRDX4 rCASP - 1 after non - reducing SDS - PAGE . Black arrows at 250 kDa indicate corresponding molecular weights of rCASP - 1 rPRDX4 oligomers decrease of Prdx4 band upon co - incubation with rCASP - 1 . Black arrow at approx . 50 kDa indicates appearance of an additional rPRDX4 band which corresponds to molecular of a Prdx4 dimer .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O

Western 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 of rPRDX4 ( = P ) rCASP - 1 ( = C ) after alkylation ( = A ) non - reducing SDS - PAGE . Black arrows indicate termination of integration of rCASP - 1 into rPRDX4 decamer / HMWC upon alkylation .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O

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 presence of non - reduced decameric rPrdx4 , reduced dimeric monomeric rPRDX4 , YVAD or control .

O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O

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 in supernatants of HEK293 transfected with plasmids for NLRP3 , ASC , IL - 1β caspase - 1 WT or Cys - to - Ser mutants C362S or C397S co - transfected with Prdx4 or control . were stimulated with 2 . 5 mM ATP for 30 min before .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O

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 of non - reducing SDS - PAGE of lysates from HEK293 transfected with caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants co - transfected with Prdx4 - GFP or GFP as control .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O

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 of co - immunoprecipitation using HA - magnetic beads from lysates of HEK293 transfected with HA - tagged caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants co - transfected with Prdx4 - GFP or GFP as control .

B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O

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.