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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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 .

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

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

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

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

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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 .

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

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 .

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 .

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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 ) .

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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 ) .

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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 .

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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 - ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 in supernatants of HEK293 transfected with plasmids for NLRP3 , ASC , IL - 1β caspase - 1 co - transfected with Prdx4 WT or Cys - to - Ala mutants C51A , C124A , C245A or DM C124A / C245A or control . were stimulated with 2 . 5 mM ATP for 30 min before .

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

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 of co - immunoprecipitation using HA - magnetic beads from lysates of HEK293 transfected with HA - tagged caspase - 1 WT or control co - transfected with Prdx4 WT or Cys - to - Ala mutants C51A , C124A , C245A or DM C124A / C245A .

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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.


Western blot of Prdx4 , pro - caspase ‑ 1 , Gapdh E - Cadherin from insoluble fraction of LPS / or ATP - stimulated BMDMs or untreated controls .

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Western 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 in supernatants of Prdx4 WT or KO BMDMs , primed for 6 h with LPS 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 - t - ) .

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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).


of Prdx4 in serum of WT mice , injected with LPS ( 4 . 5 mg / kg BW ) for time points indicated . Each dot represents an mouse . Horizontal lines indicate mean . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - t - ) .

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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).


of Prdx4 in supernatants of Prdx4 WT KO BMDMs . were primed with LPS ( 100 ng / ml ) for 6 h , followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min 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 - t - ) .

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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 of Prdx4 secretion in response to LPS + ATP stimulation pretreatment with either Glycine , NSA or GW4869 relative of caspase - 1 secretion in response to LPS + ATP stimulation pretreatment with in response to LPS + ATP stimulation 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 - t - ) .

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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 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 stimulated with 5 mM ATP for 4 h .

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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.


BCA of EV in EVs isolates . Each bar represents a mean of n = 3 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - t - ) .

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BCA analysis of EV protein concentration in EVs isolates. Each bar represents a mean of n=3 technical replicates, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Exocet of EVs particle numbers . Each bar represents a mean of n = 3 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * * , p < 0 . 001 ; n . s . not significant ( two - t - ) .

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Exocet quantification assay of EVs particle numbers. Each bar represents a mean of n=3 technical replicates, vertical lines indicate SD. *, p < 0.05; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Transmission electron microscopy ( ) of EV isolates . Three representative pictures are displayed . Scale bar indicates 200 nm .

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Transmission electron microscopy (TEM) of EV isolates. Three representative pictures are displayed. Scale bar indicates 200 nm.


of distribution of EVs . Each dot indicates diameter in nm of an vesicle . SD . * * * , p < 0 . 001 ; n . s . not significant ( one - way ANOVA , followed by a Tukey multiple comparison ) .

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Analysis of size distribution of EVs. Each dot indicates the diameter in nm of an individual vesicle. SD. ***, p < 0.001; n.s. not significant (one-way ANOVA, followed by a Tukey multiple comparison test).


Prdx4 in EV lysates . Each bar represents a mean of n = 2 technical replicates , vertical lines indicate SD . * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - t - ) .

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Prdx4 concentration in EV lysates. Each bar represents a mean of n= 2 technical replicates, vertical lines indicate SD. **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Caspase - 1 IL - 1β in EV lysates . Each bar represents a mean of n = 2 technical replicates , vertical lines indicate SD . * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - t - ) .

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Caspase-1 and IL-1β concentration in EV lysates. Each bar represents a mean of n= 2 technical replicates, vertical lines indicate SD. **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Western blot of Prdx4 , NLRP3 , ASC , caspase ‑ 1 , IL - 1β CD63 from EV lysates ( upper panel ) or caspase - 1 after immunoprecipitation against caspase - 1 ( lower panel ) .

Western blot analysis of Prdx4, NLRP3, ASC, caspase‑1, IL-1β and CD63 from EV lysates (upper panel) or caspase-1 after immunoprecipitation against caspase-1 (lower panel).


Cxcl1 in supernatants of caspase - 1 - deficient BMDMs stimulated with EVs from LPS , LPS ATP or control - treated Prdx4 WT or KO BMDMs , as well as caspase - 1 - deficient BMDMs pre - treated with Anakinra stimulated with EVs from LPS ATP - treated Prdx4 WT or KO BMDMs . Each bar represents a mean of n = 3 biological with 2 technical replicates , vertical lines indicate SD .

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Cxcl1 concentration in supernatants of caspase-1-deficient BMDMs stimulated with EVs from LPS, LPS and ATP or control-treated Prdx4 WT or KO BMDMs, as well as caspase-1-deficient BMDMs pre-treated with Anakinra and stimulated with EVs from LPS and ATP-treated Prdx4 WT or KO BMDMs. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD.


Serum Cxcl1 in C57Bl6 / N mice injected with either PBS or EVs from LPS ATP or control - treated Prdx4 WT or KO or ASC KO BMDMs . Each dot represents an mouse . Horizontal lines indicate mean .

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Serum Cxcl1 in C57Bl6/N mice injected with either PBS or EVs from LPS and ATP or control-treated Prdx4 WT or KO or ASC KO BMDMs. Each dot represents an individual mouse. Horizontal lines indicate mean.


( B ) Western blot of SARS - CoV - 2 Spike 1 - 1273 , 1 - 674 , 319 - 591 expression in HEK293T . black arrows indicate location of induced target .

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(B) Western blot analysis of SARS-CoV-2 Spike 1-1273, 1-674, and 319-591 expression in HEK293T cells. The black arrows indicate the location of the induced target proteins.


( C ) Detection of decoy antibody ACE2 - Fc Spike S1 - Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue .

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(C) Detection of decoy antibody ACE2-Fc and Spike S1-Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue staining.


( D ) Recognition of ACE2 - Fc Spike S1 - Fc by anti - human IgG Fc antibody in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( SDS - PAGE ) .

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(D) Recognition of the ACE2-Fc and Spike S1-Fc by anti-human IgG Fc antibody in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).


( E , F ) Deglycosylation of ACE2 - Fc Spike 1 - 674 - Fc by PNGase F . PNGase F digested ACE2 - Fc ( 500 ng ) Spike 1 - 674 - Fc ( 500 ng ) were subjected to Coomassie Brilliant Blue ( E ) Western blot by anti - human IgG Fc antibody ( F ) .

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(E, F) Deglycosylation of ACE2-Fc and Spike 1-674-Fc by PNGase F. PNGase F digested ACE2-Fc (500 ng) and Spike 1-674-Fc (500 ng) were subjected to Coomassie Brilliant Blue staining (E) and Western blot analysis by anti-human IgG Fc antibody (F).


( A ) Competitive ELISA . ACE2 - Fc can compete with ACE2 - Fc - Biotin for Spike 1 - 674 binding in a dose - dependent manner . competition effects of ACE2 - Fc on ACE2 - Spike binding was normalized to PBS control . Error bars represent standard deviation ( SD ) , n = 2 . Statistical was performed by unpaired two tail t - . * * P < 0 . 01 , * * * P < 0 . 001 .

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(A) Competitive ELISA. ACE2-Fc can compete with ACE2-Fc-Biotin for Spike 1-674 binding in a dose-dependent manner. The competition effects of ACE2-Fc on ACE2-Spike protein binding was normalized to PBS control. Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. **P < 0.01, ***P < 0.001.


( B ) Recognition of full - Spike on H1975 ( left panel as negative control ) or H1975 - Spike ( right panel ) by ACE2 - Fc using flow cytometry . Isotype control : 40 μg / mL mouse IgG - FITC .

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(B) Recognition of full-length Spike on H1975 (left panel as negative control) or H1975-Spike (right panel) cells by ACE2-Fc using flow cytometry analysis. Isotype control: 40 μg/mL mouse IgG-FITC.


( C ) Confocal microscopy of H1975 - Spike - overexpressing . Spikes on H1975 were stained with anti - Spike antibody Alexa Fluor ® 594 - conjugated secondary antibody on ice for 1 hr . After that , ACE2 - Fc - FITC was incubated for another 1 hr . Scale bars equal to 20 µm . DAPI was used as a counterstain .

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(C) Confocal microscopy of H1975-Spike-overexpressing cells. Spikes on H1975 cells were stained with anti-Spike antibody and Alexa Fluor® 594-conjugated secondary antibody on ice for 1 hr. After that, ACE2-Fc-FITC was incubated for another 1 hr. Scale bars equal to 20 µm. DAPI was used as a nuclear counterstain.


( D ) Preservation of ACE2 - Fc peptidase activity . peptidase activity of ACE2 - Fc was measured by cleavage of peptide substrates .

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(D) Preservation of ACE2-Fc peptidase activity. The peptidase activity of ACE2-Fc was measured by cleavage of the fluorescent peptide substrates.


( E ) Inhibition of angiotensin II ( Ang II ) - induced TNF - α production by ACE2 - Fc . Ang II was preincubated with indicated amounts of ACE2 - Fc or IgG for 30 minutes . After that , mixtures were added to RAW264 . 7 for 12 hr . of TNF - α in culture medium was determined by ELISA . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 . dotted represents mean value of TNF - α in control group .

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(E) Inhibition of angiotensin II (Ang II)-induced TNF-α production by ACE2-Fc. Ang II was preincubated with indicated amounts of ACE2-Fc or IgG for 30 minutes. After that, the mixtures were added to RAW264.7 cells for 12 hr. The concentration of TNF-α in the culture medium was determined by ELISA. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. *P < 0.05. The dotted line represents the mean value of TNF-α in control group.


( F ) Inhibition of Ang II - induced ADAM17 ( a disintegrin metalloprotease 17 ) phosphorylation by ACE2 - Fc . extracts from ( E ) were immunoblotted with indicated antibodies ( left panel ) . β - actin served as loading control . signal was normalized to only ( right panel ) . Data are representative of three independent experiments , values are expressed as mean ± SD ( right panel ) . Error bars represent standard deviation ( SD ) , n = 2 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 . IgG represents human normal IgG control .

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

(F) Inhibition of Ang II-induced ADAM17 (a disintegrin and metalloprotease 17) phosphorylation by ACE2-Fc. The protein extracts from (E) were immunoblotted with the indicated antibodies (left panel). β-actin served as the loading control. The signal intensity was normalized to cells only (right panel). Data are representative of three independent experiments, and the values are expressed as the mean ± SD (right panel). Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. *P < 0.05. IgG represents the human normal IgG control.


( B ) HEK293 H1975 were transduced with full - ACE2 by lentivirus . extracts were immunoblotted with indicated antibodies . O / E represents overexpression .

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(B) HEK293 and H1975 cells were transduced with full-length ACE2 by lentivirus. Protein extracts were immunoblotted with the indicated antibodies. O/E represents overexpression.


( C ) GFP full - Spike co - transfected HEK293T were used as effector ( 293 - S ) . HEK293 / ACE2 ( HEK293T - overexpressing ACE2 ) ( left panel ) or H1975 / ACE2 ( H1975 - overexpressing ACE2 ) ( right panel ) were used as target . 293 - S were preincubated with normal human IgG or ACE2 - Fc at 37 ° C for 1 hr . After that , mixtures were added to target for 4 hr ( - fusion ) or 24 hr ( syncytia formation ) . areas were measured by inverted microscopy Metamorph software ( Metamorphosis ) . Scale bars equal to 50 µm . arrows indicate - fusion or syncytia formation .

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

(C) GFP and full-length Spike co-transfected HEK293T cells were used as effector cells (293-S). HEK293/ACE2 (HEK293T-overexpressing ACE2) (left panel) or H1975/ACE2 (H1975-overexpressing ACE2) (right panel) cells were used as target cells. The 293-S cells were preincubated with normal human IgG or ACE2-Fc at 37°C for 1 hr. After that, the mixtures were added to target cells for 4 hr (cell-cell fusion) or 24 hr (syncytia formation). The fluorescent areas were measured by inverted fluorescence microscopy and Metamorph software (Metamorphosis). Scale bars equal to 50 µm. The white arrows indicate the cell-cell fusion or syncytia formation.


( D ) inhibition of - fusion syncytia formation by ACE2 - Fc in HEK293 / ACE2 H1975 / ACE2 was determined with formula described in Methods section . Error bars represent standard deviation ( SD ) , n = 6 . Statistical was performed by unpaired two tail t - . * * P < 0 . 01 , * * * P < 0 . 001 .

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(D) The inhibition of cell-cell fusion and syncytia formation by ACE2-Fc in HEK293/ACE2 and H1975/ACE2 was determined with the formula described in the Methods section. Error bars represent the standard deviation (SD), n=6. Statistical analysis was performed by unpaired two tail t-test. ** P < 0.01, *** P < 0.001.


( A ) ACE2 - Fc blocked entry of Spike - expressing pseudotyped lentivirus into HEK293T - ACE2 H1975 - ACE2 . relative luciferase activities , normalized to only group , represent efficiency of entry . MOI : Multiplicity of infection .

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(A) ACE2-Fc blocked the entry of Spike-expressing pseudotyped lentivirus into HEK293T-ACE2 and H1975-ACE2 cells. The relative luciferase activities, normalized to the only virus group, represent the efficiency of virus entry. MOI: Multiplicity of infection.


( B ) Confocal microscopy of airway organoids . green represents specific of indicated monoclonal antibodies Alexa Fluor 488 - conjugated secondary antibodies . DAPI was used as a counterstain . Hematoxylin eosin ( H & E ) stain : hematoxylin stained nuclei in blue color ; eosin stained cytoplasm in pink color . Scale bars equal to 20 µm .

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(B) Confocal microscopy images of the airway organoids. The green fluorescence represents the specific staining of indicated monoclonal antibodies and Alexa Fluor 488-conjugated secondary antibodies. DAPI was used as a nuclear counterstain. Hematoxylin and eosin (H&E) stain: hematoxylin stained the nuclei in blue color; eosin stained the cytoplasm in pink color. Scale bars equal to 20 µm.


( C ) Lung cancer A549 , human normal bronchial epithelial ( HBEpc ) , HBEpc - differentiated ( airway organoids ) were harvested for immunoblotting with indicated antibodies .

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(C) Lung cancer A549 cells, human normal bronchial epithelial cells (HBEpc), and HBEpc-differentiated cells (airway organoids) were harvested for immunoblotting with the indicated antibodies.


( D ) Blockage of pseudovirus entry into airway organoids by ACE2 - Fc . Mixtures of pseudotyped lentivirus with or without ACE2 - Fc were cocultured with airway organoids for 72 hr . entry was determined by quantifying luciferase activity in lysates .

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(D) Blockage of pseudovirus entry into airway organoids by ACE2-Fc. Mixtures of pseudotyped lentivirus with or without ACE2-Fc were cocultured with airway organoids for 72 hr. The virus entry was determined by quantifying the luciferase activity in the cell lysates.