d ) Immunostaining of heart sections for N2 . 261 MEF2 in 5 dpci ppargc1a + / + ppargc1a - / - ; magnified view of in boxes shown below ; arrowheads point to N2 . 261 + CMs ; dashed lines mark wound border ; of N2 . 261 + CMs in border zone shown on right ( n = 4 - 6 ventricles of each genotype ) .

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

d) Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


( e AFOG of heart sections from 60 dpci pkma2 + / - ; pkmb + / - pkma2 - / - ; pkmb + / - ; black dashed lines outline scar ; scar measured on right ( n = 3 - 5 ventricles of each genotype ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 AFOG staining of heart sections from 60 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).


f ) AFOG of heart sections from 45 dpci ppargc1a + / + ppargc1a - / - ; black dashed lines outline scar ; scar measured on right ( n = 3 - 5 ventricles of each genotype ) .

O O O O O O O O O O O O O O O O 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) AFOG staining of heart sections from 45 dpci ppargc1a+/+ and ppargc1a-/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).


Immunostaining of heart sections for PCNA MEF2 in 5 dpci ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , after tamoxifen heat shock treatments ) ; magnified view of in boxes shown below ; arrowheads point to PCNA + CMs ; dashed lines mark wound border ; of PCNA + CMs in border zone shown on right ( n = 4 - 5 ventricles of each genotype ) .

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

Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).


Immunostaining of heart sections for N2 . 261 MEF2 in 5 dpci ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , after tamoxifen heat shock treatments ) ; magnified view of in boxes shown below ; arrowheads point to N2 . 261 + CMs ; dashed lines mark wound border ; of N2 . 261 + CMs in border zone shown on right ( n = 4 - 5 ventricles of each genotype ) .

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

Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).


( f AFOG of heart sections from 60 dpci ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , after tamoxifen heat shock treatments ) ; black dashed lines outline scar ; scar measured on right ( n = 4 - 5 ventricles of each genotype ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 AFOG staining of heart sections from 60 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); black dashed lines outline the scar area; scar area measured on the right (n = 4-5 ventricles of each genotype).


g ) AFOG of heart sections from 60 dpci Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , after tamoxifen heat shock treatments ) ; black dashed lines outline scar ; scar measured on right ( n = 4 - 5 ventricles of each genotype ) .

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

g) AFOG staining of heart sections from 60 dpci animals and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); black dashed lines outline the scar area; scar area measured on the right (n = 4-5 ventricles of each genotype).


( a , b ) for Ki67 , CTNI ( DAPI ) in control , PDHA1STA OE or PDK3 OE RNCMs cultured in growth ( a ) or non - growth ( b ) medium ; arrowheads point to Ki67 + RNCMs ; of Ki67 + RNCMs shown on right ( n = 4 biological replicates for each condition ) .

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

(a, b) Staining for Ki67, CTNI and DNA (DAPI) in control, PDHA1STA OE or PDK3 OE RNCMs cultured in growth (a) or non-growth (b) medium; arrowheads point to Ki67+ RNCMs; percentage of Ki67+ RNCMs shown on the right (n = 4 biological replicates for each condition).


( c ) for CTNI ( DAPI ) in scratch using control , PDHA1STA OE or PDK3 OE RNCMs immediately or at 3 days after generating scratch ; magnified view of in boxes shown below ; arrowheads point to protrusions of RNCMs ; of CM protrusions shown on right ( n = 4 biological replicates for each condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Staining for CTNI and DNA (DAPI) in scratch assay using control, PDHA1STA OE or PDK3 OE RNCMs immediately or at 3 days after generating the scratch; magnified view of area in white boxes shown below; arrowheads point to membrane protrusions of RNCMs; quantification of CM membrane protrusions shown on the right (n = 4 biological replicates for each condition).


( d ) Reactome over - representation of differentially regulated genes in PDK3 OE RNCMs compared to control .

(d) Reactome over-representation analysis of differentially regulated genes in PDK3 OE RNCMs compared to control.


( e Heat map of gene expression for key regulators of cycle in PDK3 OE vs control RNCMs .

(e Heat map of gene expression for key regulators of cell cycle in PDK3 OE vs control RNCMs.


f ) Heat map of gene expression for key regulators of replication in PDK3 OE vs control RNCMs .

f) Heat map of gene expression for key regulators of DNA replication in PDK3 OE vs control RNCMs.


( a ) H3R expression in cultured ratcortical neurons at DIV7 after OGD / R injury ( 2 h OGD 24 h reperfusion ) shown by western blot ( n = 6 per condition ; * * P0 . 01 versus control with Student ' s t - ) . Full - blots are shown in Supplementary Fig . 9 .

(a) H3R expression in cultured ratcortical neurons at DIV7 after OGD/R injury (2 h OGD and 24 h reperfusion) shown by western blot (n=6 per condition; **P0.01 versus control with Student's t-test). Full-size blots are shown in Supplementary Fig. 9.


( b ) When H3R antagonist thioperamide ( THIO , 10 − 6 mol l − 1 at reperfusion ) agonist immepip ( IMME , 10 − 6 mol l − 1 at reperfusion ) were administered , viability was tested in cultured neurons by 3 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 2 , 5 - diphenyltetrazolium bromide ( MTT ) after OGD / R ( n = 7 per condition ; * * * P0 . 001 with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc ) .

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) When the H3R antagonist thioperamide (THIO, 10−6 mol l−1 at reperfusion) and the agonist immepip (IMME, 10−6 mol l−1 at reperfusion) were administered, viability was tested in cultured neurons by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after OGD/R (n=7 per condition; ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( c ) 2 , 3 , 5 - triphenyltetrazolium chloride - stained brain sections showing infarct in WT H3R − / − mice receiving saline , H3R antagonists A331440 , clobenpropit ( CLOB ) thioperamide , ( THIO ; 5 mg kg − 1 , i . p . , immediately at reperfusion 6 h later ) , H3R agonist immepip ( IMME , 1 μg , i . c . v . , at reperfusion ) after I / R ( 1 h MCAO followed by 24 h reperfusion ; scale bar , 5 mm ) .

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

(c) 2,3,5-triphenyltetrazolium chloride-stained brain sections showing the infarct area in WT and H3R−/− mice receiving saline, the H3R antagonists A331440, clobenpropit (CLOB) and thioperamide, (THIO; 5 mg kg−1, i.p., immediately at reperfusion and 6 h later), and the H3R agonist immepip (IMME, 1 μg, i.c.v., at reperfusion) after I/R (1 h MCAO followed by 24 h reperfusion; scale bar, 5 mm).


bar graphs show infarct volume ( d ) neurological score ( e ) ( n = 6 - 8 mice per condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 versus WT saline group with ANOVAs followed by Bonferroni / Dunn post hoc ) . Data are presented as mean ± s . e . m .

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

The bar graphs show infarct volume (d) and neurological score (e) (n=6-8 mice per condition, *P0.05, **P0.01, ***P0.001 versus WT saline group with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) TUNEL of apoptotic in penumbral brain slices in vivo ( scale bar , 100 μm ) when H3R was inhibited with thioperamide ( scale bar , 50 μm ) after tMCAO . TUNEL - positive are green , are stained with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) .

O 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

(a) TUNEL staining of apoptotic cells in penumbral brain slices in vivo (scale bar, 100 μm) when H3R was inhibited with thioperamide (scale bar, 50 μm) after tMCAO. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).


( b , d ) H3R was inhibited by thioperamide , caspase - 3 expression is shown by western blot in WT H3R − / − mice after tMCAO ( b ) ( n = 6 - 7 per condition ; * * P0 . 01 , * * * P0 . 001 with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc )

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

(b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice after tMCAO (b) (n=6-7 per condition; **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by Bonferroni/Dunn post hoc test)


( c ) TUNEL of apoptotic in cultured neurons when H3R was inhibited with thioperamide ( scale bar , 50 μm ) after OGD / R . TUNEL - positive are green , are stained with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) .

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

(c) TUNEL staining of apoptotic cells in cultured neurons when H3R was inhibited with thioperamide (scale bar, 50 μm) after OGD/R. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).


( b , d ) H3R was inhibited by thioperamide , caspase - 3 expression is shown by western blot in WT H3R − / − mice in cultured neurons subjected to OGD / R ( d ) ( n = 8 per condition ; * P0 . 05 , * * * P0 . 001 with ANOVAs followed by Bonferroni / Dunn post hoc ) . Data are presented as mean ± s . e . m . Full - blots are shown in Supplementary Fig . 10 .

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

(b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice in cultured neurons subjected to OGD/R (d) (n=8 per condition; *P0.05, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 10.


( a ) 2 , 3 , 5 - Triphenyltetrazolium chloride ( TTC ) - stained brain sections from WT HDC − / − mice showing infarct in those receiving saline , H3R antagonist thioperamide ( THIO ) , H1R antagonist pyrilamine ( PYRI ; 10 mg kg − 1 , i . p . , at reperfusion ) , H2R antagonist cimetidine ( CIME ; 10 mg kg − 1 , i . p . , at reperfusion ) α - FMH ( 50 mg kg − 1 , i . p . , 2 h before ischemia ) after 24 h reperfusion ( n = 6 per condition ; scale bar , 5 mm ) .

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

(a) 2,3,5-Triphenyltetrazolium chloride (TTC)-stained brain sections from WT and HDC−/− mice showing the infarct area in those receiving saline, the H3R antagonist thioperamide (THIO), the H1R antagonist pyrilamine (PYRI; 10 mg kg−1, i.p., at reperfusion), the H2R antagonist cimetidine (CIME; 10 mg kg−1, i.p., at reperfusion) and α-FMH (50 mg kg−1, i.p., 2 h before ischemia) after 24 h reperfusion (n=6 per condition; scale bar, 5 mm).


( b ) Infarct volumes ( c ) neurological deficit scores ( * P0 . 05 , * * P0 . 01 , * * * P0 . 001 with of variances followed by Bonferroni / Dunn post hoc ) . Data are presented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Infarct volumes and (c) neurological deficit scores (*P0.05, **P0.01, ***P0.001 with analysis of variances followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) Akt / GSK - 3β / mTOR / P70S6K phosphorylation assessed by western blots after thioperamide administration OGD / R . LiCl ( 5 mmol l − 1 immediately at reperfusion ) was used to rephosphorylate GSK - 3β / mTOR / P70S6K . digits below represent semiquantified optidensity of bands , control bands were defined as 1 . 00 .

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 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

(a) Akt/GSK-3β/mTOR/P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD/R. LiCl (5 mmol l−1 immediately at reperfusion) was used to rephosphorylate GSK-3β/mTOR/P70S6K. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.


( b ) Representative western blots showing phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signaling in H3R − / − mice after OGD / R injury . digits below represent semiquantified optidensity of bands , control bands were defined as 1 . 00 .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blots showing the phosphorylation of Akt/GSK-3β/mTOR/P70S6K signaling in H3R−/− mice after OGD/R injury. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.


( c ) Representative showing LC3 - positive autophagic vacuoles ( red ) with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) after administration of thioperamide LiCl with OGD / R ( scale bar , 10 μm ) .

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

(c) Representative images showing LC3-positive autophagic vacuoles (red) with 4',6-diamidino-2-phenylindole staining (blue) after administration of thioperamide and LiCl with OGD/R (scale bar, 10 μm).


( d ) viability assessed by MTT when thioperamide LiCl were administered after OGD / R ( n = 7 , * * P0 . 01 , * * * P0 . 001 with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc ) .

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

(d) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD/R (n=7, **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( e ) Representative western blots bar graph showing expression of LC3 - I LC3 - II in steady - state autophagy autophagic flux under OGD / R injury when thioperamide ( THIO ) immepip ( IMME ) were administered ( n = 6 ; * * P0 . 01 , * * * P0 . 001 with ANOVAs followed by Bonferroni / Dunn post hoc ) . Data are presented as mean ± s . e . m . Full - blots are shown in Supplementary Fig . 11 .

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

(e) Representative western blots and bar graph showing the expression of LC3-I and LC3-II in steady-state autophagy and autophagic flux under OGD/R injury when thioperamide (THIO) and immepip (IMME) were administered (n=6; **P0.01, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 11.


( a ) Representative immunohistochemical of LC3 ( green ) along with NeuN ( red ) in WT H3R − / − mice ( scale bar , 25 μm ) .

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

(a) Representative immunohistochemical staining of LC3 (green) along with NeuN staining (red) in WT and H3R−/− mice (scale bar, 25 μm).


( b , c ) When thioperamide was administered in vivo , LC3 expression was assessed by western blot after I / R in WT mice ( b ) ( * P0 . 05 , * * * P0 . 001 ; n = 6 mice per condition , with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc ) , in H3R − / − mice ( c ) ( * * * P0 . 001 versus H3R − / − sham group ; n = 5 - 6 mice per condition , with ANOVAs followed by Bonferroni / Dunn post hoc . ) Data are presented as mean ± s . e . m . Full - blots are shown in Supplementary Fig . 12 .

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

(b,c) When thioperamide was administered in vivo, LC3 expression was assessed by western blot after I/R in WT mice (b) (*P0.05, ***P0.001; n=6 mice per condition, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test), and in H3R−/− mice (c) (***P0.001 versus H3R−/− sham group; n=5-6 mice per condition, with ANOVAs followed by the Bonferroni/Dunn post hoc test.) Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 12.


( a ) Western blots showing inhibitory effect of 3 - MA on autophagy in I / R . Full - blots are shown in Supplementary Fig . 13 .

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

(a) Western blots showing the inhibitory effect of 3-MA on autophagy in I/R. Full-size blots are shown in Supplementary Fig. 13.


( b ) TTC - stained sections showing infarct after administration of 3 - MA ( 100 nmol , i . c . v . , at reperfusion ) in WT H3R − / − mice ( scale bar , 5 mm ) , ( c ) bar graph showing infarct volume ( n = 6 - 8 mice per condition ; * P0 . 05 , * * * P0 . 001 , with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TTC-stained sections showing the infarct area after administration of 3-MA (100 nmol, i.c.v., at reperfusion) in WT and H3R−/− mice (scale bar, 5 mm), and (c) bar graph showing infarct volume (n=6-8 mice per condition; *P0.05, ***P0.001, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( d ) TTC - stained sections ( scale bar , 5 mm ) bar graph showing infarct after administration of thioperamide in Atg5 + / − mice ( n = 4 - 6 mice per condition ) .

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

(d) TTC-stained sections (scale bar, 5 mm) and bar graph showing the infarct area after administration of thioperamide in Atg5+/−mice (n=4-6 mice per condition).


( e ) In cultured neurons , viability was tested by MTT after 3 - MA ( 2 . 5 mmol l − 1 at reperfusion ) thioperamide was administered in OGD / R ( n = 7 per condition ; * * * P0 . 001 , with ANOVAs followed by Bonferroni / Dunn post hoc ) .

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

(e) In cultured neurons, viability was tested by MTT assay after 3-MA (2.5 mmol l−1 at reperfusion) and thioperamide was administered in OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


( f ) In cultured neurons , viability was tested by MTT after siRNA for Atg7 OGD / R ( n = 7 per condition ; * * * P0 . 001 , with ANOVAs followed by Bonferroni / Dunn post hoc ) .

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

(f) In cultured neurons, viability was tested by MTT after siRNA for Atg7 and OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


( g ) Viability was tested by MTT on Atg5 + / + Atg5 − / − MEFs after thioperamide administration after OGD / R ( n = 7 per condition ; * * P0 . 01 , * * * P0 . 001 , with ANOVAs followed by Bonferroni / Dunn post hoc ) . Data are presented as mean ± s . e . m .

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(g) Viability was tested by MTT assay on Atg5+/+and Atg5−/− MEFs after thioperamide administration after OGD/R (n=7 per condition; **P0.01, ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) Representative co - immunoprecipitation results showing effect of thioperamide on interaction of H3R with CLIC4 during OGD / R . Full - blots are shown in Supplementary Fig . 14 .

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(a) Representative co-immunoprecipitation results showing the effect of thioperamide on the interaction of H3R with CLIC4 during OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( b ) viability was tested by MTT after administration of Tat - H3RCT414 - 436 ( 10 − 6 mol l − 1 at reperfusion ) under OGD / R ( data are presented as mean ± s . e . m . ; n = 7 per condition ; * P0 . 05 , * * P0 . 01 , with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc ) .

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(b) Cell viability was tested by MTT assay after administration of Tat-H3RCT414-436 (10−6 mol l−1 at reperfusion) under OGD/R (data are presented as mean±s.e.m.; n=7 per condition; *P0.05, **P0.01, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( c ) Representative western blots showing effect of Tat - H3RCT414 - 436 on phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signalling LC3 expression in OGD / R . Full - blots are shown in Supplementary Fig . 14 .

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(c) Representative western blots showing the effect of Tat-H3RCT414-436 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( d ) Representative of effect of Tat - H3RCT414 - 436 on LC3 - positive autophagic ( red ) in 4 ' , 6 - diamidino - 2 - phenylindole - stained ( blue ) neurons after OGD / R ( scale bar , 10 μm ) .

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(d) Representative images of the effect of Tat-H3RCT414-436 on LC3-positive autophagic puncta (red) in 4',6-diamidino-2-phenylindole-stained (blue) neurons after OGD/R (scale bar,10 μm).


( e ) Western blots showing effect of siRNA for CLIC4 on phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signalling LC3 expression in OGD / R . Full - blots are shown in Supplementary Fig . 14 .

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(e) Western blots showing the effect of siRNA for CLIC4 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( f ) Representative western blots bar graph showing expression of LC3 - I LC3 - II on steady - state autophagy autophagic flux under OGD / R when CLIC4 was knocked down ( data are presented as mean ± s . e . m . ; n = 6 ; * P0 . 05 , * * P0 . 01 ; , with ANOVAs followed by Bonferroni / Dunn post hoc ) . Full - blots are shown in Supplementary Fig . 14 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blots and bar graph showing the expression of LC3-I and LC3-II on steady-state autophagy and autophagic flux under OGD/R when CLIC4 was knocked down (data are presented as mean±s.e.m.; n=6; *P0.05, **P0.01; , with ANOVAs followed by the Bonferroni/Dunn post hoc test). Full-size blots are shown in Supplementary Fig. 14.


( g ) viability was assessed by MTT after siRNA for CLIC4 under OGD / R ( data are presented as mean ± s . e . m . ; n = 8 per condition ; * * * P0 . 001 , with ANOVAs followed by Bonferroni / Dunn post hoc ) .

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

(g) Cell viability was assessed by MTT assay after siRNA for CLIC4 under OGD/R (data are presented as mean±s.e.m.; n=8 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


Immunoblot showed HEK293 transfected for 48 hours with expression vectors for TCHP , FLAG - TCHP at N - C - terminal . total lysates were immunoprecipitated with anti - FLAG antibodies , blots were probed sequentially with anti - PCM1 , anti - FLAG , anti - ACTIN antibodies . IgG chains are indicated with Red Ponceau . input totals were analyzed by parallel immunoblotting as a control for level of expression .

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

Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP, and FLAG-TCHP at N- and C- terminal. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.


Immunoblot showed HEK293 transfected for 48 hours with expression vectors for TCHP - FLAG C - terminal or constructs with deletions of coiled - coil domain 1 ( TCHP ∆ 1 ) 2 ( TCHP ∆ 2 ) , as indicated in scheme . total lysates were immunoprecipitated with anti - FLAG antibodies , blots were probed sequentially with anti - PCM1 , anti - FLAG , anti - ACTIN antibodies . IgG chains are indicated with Red Ponceau . input totals were analyzed by parallel immunoblotting as a control for level of expression .

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

Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP-FLAG C-terminal or constructs with deletions of the coiled-coil domain 1 (TCHP ∆1) and 2 (TCHP ∆2), as indicated in the scheme. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.


Anti - PCM1 immunoprecipitation from HUVECs TCHP PCM1 immunoblot .

Anti-PCM1 immunoprecipitation from HUVECs cells and TCHP and PCM1 immunoblot.


Co - localization of TCHP - V5 centriolar satellite . HUVECs were transduced with TCHP - V5 lentivirus stained for anti - PCM1 , anti - CEP290 , anti - CEP72 anti - V5 antibodies . Scale bars , 25 µm 5 µm in inset . Right panel : two - channel correlation of pixels corresponding to regions identified with TCHP - V5 satellite markers ( n = 90 ; Pearson colocalization coefficient ) .

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

Co-localization of TCHP-V5 and centriolar satellite proteins. HUVECs were transduced with TCHP-V5 lentivirus and stained for anti-PCM1, anti-CEP290, anti-CEP72 and anti-V5 antibodies. Scale bars, 25 µm and 5 µm in the inset. Right panel: the two-channel intensity correlation of pixels corresponding to regions identified with TCHP-V5 and satellite markers (n=90 cells; Pearson colocalization coefficient).


TCHP knock - down or control were stained for anti - PCM1 antibody . Panel below : ( n = 50 ; unpaired t - ; * * p = 0 . 004 vs control ) . Scale bars , 50 µm 5 µm in inset .

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

TCHP knock-down or control cells were stained for anti-PCM1 antibody. Panel below: quantification (n=50 cells; unpaired t-test; **p=0.004 vs control). Scale bars, 50 µm and 5 µm in the inset.


Control or TCHP knock - down HUVECs were subjected to cycloheximide ( CHX ) MG132 treatments for indicated number of hours before immunoblotting . PCM1 were analysed in insoluble fraction . Laminin has been used as a loading control . Below panel : of PCM1 degradation . ( one - way ANOVA ; * p = 0 . 0180 vs control time 0 ) .

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Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. PCM1 levels were analysed in the insoluble fraction. Laminin has been used as a loading control. Below panel: Quantification of PCM1 degradation. (one-way ANOVA; *p=0.0180 vs control time 0).


Control or TCHP knock - down HUVECs were subjected to cycloheximide ( CHX ) MG132 treatments for indicated number of hours before immunoblotting . Condition as in ( A ) , Western blot for anti - GABARAP anti - ACTIN antibodies . Below panel : of GABARAP degradation . ( one - way ANOVA ; * p = 0 . 0215 vs control time 0 ) .

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Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. Condition as in (A), Western blot for anti-GABARAP and anti-ACTIN antibodies. Below panel: Quantification of GABARAP degradation. (one-way ANOVA; *p =0.0215 vs control time 0).


Control or TCHP overexpressing HUVECs were subjected to CHX treatment for indicated number of hours prior to immunoblotting . Western blot was probed for anti - PCM1 anti - V5 antibodies . Laminin has been used as a loading control .

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Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-PCM1 and anti-V5 antibodies. Laminin has been used as a loading control.


Control or TCHP overexpressing HUVECs were subjected to CHX treatment for indicated number of hours prior to immunoblotting . Western blot was probed for anti - V5 , anti - GABARAP anti - ACTIN antibodies . Below panels : of C ( one - way ANOVA ; * * p < 0 . 0001 vs control time 0 ; # # p = 0 . 0003 vs control CHX ) D ( one - way ANOVA ; * * p < 0 . 0001 vs control time 0 ; # # p = 0 . 0056 vs control CHX ) .

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

Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-V5, anti-GABARAP and anti-ACTIN antibodies. Below panels: Quantification of C (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0003 vs control CHX) and D (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0056 vs control CHX).


Immunofluorescent for LC3 p62 in TCHP knock - down control . Scale bars , 25μm . Lower panel : . ( n = 90 , unpaired t - ; LC3 : * * p < 0 . 0001 vs control ; p62 : * * p < 0 . 0001 vs control ) .

Immunofluorescent staining for LC3 and p62 in TCHP knock-down and control cells. Scale bars, 25μm. Lower panel: quantification. (n=90 cells, unpaired t-test; LC3: **p<0.0001 vs control; p62: **p<0.0001 vs control).


Western blot of p62 LC3 during under normal culture condition or starved condition ( HBSS ) or presence of BafA1 ( 2 hours ) in TCHP knock - down or control . Lower panels : p62 . ( one - way ANOVA ; * * p = 0 . 0003 vs control , # # p = 0 . 0093 vs shTCHP ) ; LC3 . ( one - way ANOVA ; * * p = 0 . 0008 vs control , # # p = 0 . 0011 vs shTCHP ) .

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Western blot of p62 and LC3 during under normal culture condition or starved condition (HBSS) or the presence of BafA1 (2 hours) in TCHP knock-down or control cells. Lower panels: p62 quantification. (one-way ANOVA; **p=0.0003 vs control, ##p=0.0093 vs shTCHP); LC3 quantification. (one-way ANOVA; **p=0.0008 vs control, ##p=0.0011 vs shTCHP).


HUVECs were transduced with tandem mCherry - EGFP - LC3 with shRNA TCHP or control vectors . Left panels : Representative picture of mCherrry - EGFP - LC3 reporters . Scale bars , 25μm . Right panel : of number of mCherry - only ( red bars , autolysosomes ) or double - positive ( mCherry + / EGFP + ; yellow bars , autophagosomes ) . ( n = 80 , one - way ANOVA ; * p = 0 . 0447 vs control ; # # p = 0 . 0007 vs shTCHP ) .

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HUVECs were transduced with the tandem mCherry-EGFP-LC3 and with shRNA TCHP or control vectors. Left panels: Representative picture of mCherrry-EGFP-LC3 reporters. Scale bars, 25μm. Right panel: Quantification of the number of mCherry-only (red bars, autolysosomes) or double-positive (mCherry+/EGFP+; yellow bars, autophagosomes). (n=80 cells, one-way ANOVA; *p=0.0447 vs control; ##p=0.0007 vs shTCHP).


Ratiometric flow cytometric of mCherrry - EGFP - LC3 reporters as in C . ( one - way ANOVA ; * p = 0 . 0247 vs control ; # # p = 0 . 0088 vs shTCHP ) .

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Ratiometric flow cytometric analysis of mCherrry-EGFP-LC3 reporters as in C. (one-way ANOVA; *p=0.0247 vs control; ##p=0.0088 vs shTCHP).


of long - lived degradation in TCHP knock - down control by flow cytometry ( one - way ANOVA ; * * p = 0 . 0018 vs control , # # p < 0 . 0001 vs shTCHP ) .

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

Quantification of long-lived protein degradation assay in TCHP knock-down and control cells by flow cytometry (one-way ANOVA; **p=0.0018 vs control, ##p<0.0001 vs shTCHP).


HUVECs were transduced with TCHP - V5 control vectors . Western blot for anti - V5 anti - p62 antibody during under normal culture condition or HBSS or BafA1 or Torin - 1 treatment . Lower panel : . ( one - way ANOVA ; * * p = 0 . 0044 ; # p = 0 . 0117 vs TCHP - V5 ) .

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HUVECs were transduced with TCHP-V5 and control vectors. Western blot for anti-V5 and anti-p62 antibody during under normal culture condition or HBSS or BafA1 or Torin-1 treatment. Lower panel: quantification. (one-way ANOVA; **p=0.0044; #p=0.0117 vs TCHP-V5).


postnuclear fraction ( PNS ) from TCHP knock - down control HUVECs in presence or absence of BafA1 was separated into ( low - speed pellet ) LSP ( high - speed pellet ) HSP fractions then analysed by immunoblots using anti p62 , LC3 GABARAP antibodies . sub - fractions were treated with proteinase K ( Prot . K ) with or without Triton X - 100 ( TX - 100 ) . of Western blot : p62 , LC3 GABARAP relative to respective GAPDH were quantified using densitometry normalized to value of non - treated samples .

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

The postnuclear fraction (PNS) from TCHP knock-down and control HUVECs in the presence or absence of BafA1 was separated into (low-speed pellet) LSP and (high-speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies. The sub-fractions were treated with proteinase K (Prot. K) with or without Triton X- 100 (TX-100). Quantification of the Western blot: the p62, LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non-treated samples.


Representative of autophagosomes in control TCHP knock - down HUVECs in complete medium after 3h incubation with BafA1 ( Scale bars , 600nm ) : i , of mean autophagic vesicles ( AVs ) autophagosome ( APs ) ii , distribution of cross - section areas of analysed vesicles expressed in . ( n = 7 ; > 100 vesicles per sample ; AVs : unpaired t - ; * * p < 0 . 0001 vs control ; APs : unpaired t - * * p < 0 . 0001 vs control ) .

Representative TEM images of autophagosomes in control and TCHP knock-down HUVECs in the complete medium after 3h incubation with BafA1 (Scale bars, 600nm): i, quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and ii, distribution of the cross-section areas of the analysed vesicles expressed in percentage. (n=7 cells; >100 vesicles per sample; AVs: unpaired t-test; **p<0.0001 vs control; APs: unpaired t-test **p<0.0001 vs control).


Left panels : Representative single - channel merged of HUVECs expressing GFP - LC3 immunostained for STX17 . Scale bars , 25 µm 2 µm in inset . Right panels : of LC3 / STX17 positive in presence or absence of BafA1 ( one - way ANOVA ; * * p = 0 . 0060 vs control ; # p = 0 . 0136 vs control BafA1 ) LC3 / STX17 positive per ( n = 50 ; one - way ANOVA ; * * p < 0 . 0001 vs shTCHP BafA1 ) .

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

Left panels: Representative single-channel and merged images of HUVECs expressing GFP-LC3 and immunostained for STX17. Scale bars, 25 µm and 2 µm in the inset. Right panels: Quantification of LC3/STX17 positive cells in the presence or absence of BafA1 (one-way ANOVA; **p=0.0060 vs control; #p=0.0136 vs control BafA1) and quantification LC3/STX17 positive puncta per cells (n=50 cells; one-way ANOVA; **p<0.0001 vs shTCHP BafA1).


Expression of TCHP , p62 , IL - 1β , IL - 8 , IL - 6 in ECs from vessels wall from with CAD ( n = 8 per group , unpaired t - ; TCHP * * p < 0 . 0001 ; p62 * * p = 0 . 0007 ; IL - 1β * * p < 0 . 0001 ; IL - 8 * * p < 0 . 0001 ; IL - 6 * * p < 0 . 0001 vs . Healthy subject ) .

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Expression of TCHP, p62, IL-1β, IL-8, and IL-6 in ECs from vessels wall from patients with CAD (n=8 patients per group, unpaired t-test; TCHP **p <0.0001; p62 **p = 0.0007; IL-1β **p <0.0001; IL-8 **p <0.0001; and IL-6 **p <0.0001 vs. Healthy subject).


, of p62 in ECs from healthy subject either treated with vehicle ( DMSO ) or BAY . Scale bars , 50 µm ; ( n = 3 per group ; n = 80 ; one - way ANOVA ; * * p < 0 . 0001 vs healthy DMSO ; # # p = 0 . 003 vs DMSO ) .

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

Staining, and quantification of p62 in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. Scale bars, 50 µm; (n=3 patients per group; n=80 cells; one-way ANOVA; **p<0.0001 vs healthy DMSO; ##p=0.003 vs patients DMSO).


Expression of IL - 6 , IL - 8 IL - 1β ( n = 3 per group ; one - way ANOVA ; IL - 6 : * p = 0 . 0135 vs healthy DMSO ; # p = 0 . 0180 vs DMSO ; IL - 8 : * p = 0 . 0139 vs healthy DMSO ; # # p = 0 . 0024 vs DMSO ; IL - 1β : * p = 0 . 0104 vs healthy DMSO ; # # p = 0 . 0069 vs DMSO ) .

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Expression of IL-6, IL-8 and IL-1β (n=3 patients per group; one-way ANOVA; IL-6: *p = 0.0135 vs healthy DMSO; #p = 0.0180 vs patients DMSO; IL-8: *p = 0.0139 vs healthy DMSO; ##p = 0.0024 vs patients DMSO; IL-1β: *p = 0.0104 vs healthy DMSO; ##p = 0.0069 vs patients DMSO).


migration speed , in ECs from healthy subject either treated with vehicle ( DMSO ) or BAY . ( n = 3 per group ; one - way ANOVA ; * p = 0 . 0325 vs healthy DMSO ; # p = 0 . 0336 vs DMSO ) .

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

migration speed, in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. (n=3 patients per group; one-way ANOVA; *p=0.0325 vs healthy DMSO; #p=0.0336 vs patients DMSO).


Left panels : representative of heart of wild - type Tchp knock - out mice stained for CD31 ( green ) p62 ( magenta ) . Scale bars , 100µm , 50µm for inset . Right panels : of of cardiac ( n = 4 mice per group ; unpaired t - ; * * p = 0 . 083 vs wild - type ) or vessels positive for p62 ( n = 4 mice per group ; unpaired t - ; * * p = 0 . 058 vs wild - type ) vessel density ( n = 4 mice per group ; unpaired t - ; * * p = 0 . 0097 vs wild - type ) .

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

Left panels: representative images of the heart of wild-type and Tchp knock-out mice stained for CD31 (green) and p62 (magenta). Scale bars, 100µm, and 50µm for the inset. Right panels: Quantification of the percentage of cardiac area (n=4 mice per group; unpaired t-test; **p=0.083 vs wild-type) or vessels positive for p62 (n=4 mice per group; unpaired t-test; **p=0.058 vs wild-type) and vessel density (n=4 mice per group; unpaired t-test; **p=0.0097 vs wild-type).


Electron micrograph of fibrils formed by a synthetic peptide encompassing M45 RHIM . Representative of three samples examined . Scale bar 100

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

Electron micrograph of fibrils formed by a synthetic peptide encompassing the M45 RHIM. Representative of three samples examined. Scale bar 100 nm


HT29 survival 18h after treatment with TNF + BV - 6 + z - VAD - fmk to induce necroptosis . expressed lentivirus constructs encoding 1 - 90 or 1 - 277 fragments of M45 were cultured + / - addition of coumermycin A1 to induce dimerisation . N = 2 for M45 - 90 gyrase + / - coumermycin A1 4 for M45 - 277 gyrase for empty vector , + / - coumermycin

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

HT29 cell survival 18h after treatment with TNF+BV-6+z-VAD-fmk to induce necroptosis. Cells expressed lentivirus constructs encoding 1-90 or 1-277 fragments of M45 and were cultured +/- the addition of coumermycin A1 to induce dimerisation. N=2 for M45-90 gyrase +/- coumermycin A1 and 4 for M45-277 gyrase and for empty vector, +/- coumermycin A1


Thioflavin T as a function of time , following dilution of WT or AAAA His ‑ Ub ‑ M451 ‑ 90 , His - Ub - RIPK1497 ‑ 583 , His - Ub - RIPK3387 ‑ 518 from 8 M to 200 mM urea - containing buffer . Buffer sample contains ThT but no . Samples were tested in triplicate solid is average of triplicates error bars ( in grey ) represent + / - s .

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Thioflavin T fluorescence as a function of time, following dilution of WT or AAAA His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 from 8 M to 200 mM urea-containing buffer. Buffer sample contains ThT but no protein. Samples were tested in triplicate and solid line is the average of the triplicates and the error bars (in grey) represent +/- s.d


Absorbance spectra from solutions containing Congo red with WT ( black lines ) or AAAA mutant ( grey lines ) His ‑ Ub ‑ M451 ‑ 90 , His - Ub - RIPK1497 ‑ 583 , His - Ub - RIPK3387 ‑ 518 that have been dialysed against Assembly Buffer . Congo red sample alone with no ( grey ) served as a control for experiment . Experiment conducted

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

Absorbance spectra from solutions containing Congo red with WT (black lines) or AAAA mutant (dark grey lines) His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 that have been dialysed against Assembly Buffer. Congo red sample alone with no protein (light grey line) served as a control for the experiment. Experiment conducted once


Electron micrograph of material eluted in peak 1 from three MBP fusion , as labelled . These are representative of material observed . Scale bar 100 nm in

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Electron micrograph of material eluted in peak 1 from the three MBP fusion proteins, as labelled. These are representative micrographs of the material observed. Scale bar 100 nm in all panels


Representative of insoluble material generated after treatment with thrombin removed MBP . Experiment repeated three times . Scale bar 100 nm in

Representative micrographs of insoluble material generated after treatment with thrombin removed the MBP. Experiment repeated three times. Scale bar 100 nm in all panel


polarisation profiles from incorporation of FITC - M451 ‑ 90 into excess unlabeled M451 ‑ 90 ( green ) , RIPK1497 ‑ 583 ( red ) , RIPK3387 ‑ 518 ( blue ) or ZBP1170 - 355 ( purple ) . Average polarisation profile from smoothed triplicate samples plotted , with s . d . indicated . Experiment performed twice representative results shown

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

Fluorescence polarisation profiles from incorporation of FITC-M451‑90 into excess unlabeled M451‑90 (green), RIPK1497‑583 (red), RIPK3387‑518 (blue) or ZBP1170-355 (purple). Average polarisation profile from smoothed triplicate samples plotted, with s.d. indicated. Experiment performed twice and representative results shown here


Representative trace showing homomeric assembly over 40 s in mixtures of ( C ) mCHERRY - RIPK1497 ‑ 583 YPet - RIPK1497 ‑ 583 ( D ) mCHERRY - RIPK3387 ‑ 518 YPet - RIPK3387 ‑ 518 . E , F Representative traces showing heteromeric assembly over 40 s in mixtures of ( E ) mCHERRY - RIPK1497 ‑ 583 YPet - RIPK3387 ‑ 518 ( inset is expansion of region indicated by * ) ( F ) mCHERRY - RIPK1497 ‑ 583 - AAAA YPet - RIPK3387 ‑ 518 . mixtures examined in at least three different experiments on three separate

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

Representative fluorescence intensity trace showing homomeric assembly over 40 s in mixtures of (C) mCHERRY-RIPK1497‑583 and YPet-RIPK1497‑583 and (D) mCHERRY-RIPK3387‑518 and YPet-RIPK3387‑518. E, F Representative fluorescence intensity traces showing heteromeric assembly over 40 s in mixtures of (E) mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 (inset is expansion of region indicated by *) and (F) mCHERRY-RIPK1497‑583-AAAA and YPet-RIPK3387‑518. All mixtures examined in at least three different experiments on three separate occasions


Representative traces collected over 50 s for mixtures of ( A ) mCHERRY - M451 - 90 YPet ‑ only ; ( B ) mCHERRY - M451 - 90 YPet ‑ RIPK1497 ‑ 583 ; ( C ) mCHERRY - M451 - 90 YPet - RIPK3387 ‑ 518 ; ( D ) mCHERRY - M451 - 90 YPet - ZBP1170 - 355 ; ( E ) mCHERRY - M451 - 90AAAA YPet ‑ only ; ( F ) mCHERRY - M451 - 90AAAA YPet - RIPK1497 - 583 ; ( G ) mCHERRY - M451 - 90AAAA YPet - RIPK3387 ‑ 518 ( H ) mCHERRY - M451 - 90 YPet - ZBP1170 - 355 . Independent replicates performed at least

Representative fluorescence intensity traces collected over 50 s for mixtures of (A) mCHERRY-M451-90 and YPet‑only; (B) mCHERRY-M451-90 and YPet‑RIPK1497‑583; (C) mCHERRY-M451-90 and YPet-RIPK3387‑518; (D) mCHERRY-M451-90 and YPet-ZBP1170-355; (E) mCHERRY-M451-90AAAA and YPet‑only; (F) mCHERRY-M451-90AAAA and YPet-RIPK1497-583; (G) mCHERRY-M451-90AAAA and YPet-RIPK3387‑518 and (H) mCHERRY-M451-90 and YPet-ZBP1170-355. Independent replicates performed at least twice


Representative electron of homofibrils formed by M451 - 90 , RIPK1497 ‑ 583 , His - Ub - RIPK3387 ‑ 518 or ZBP1170 - 355 individually , heterofibrils formed by indicated mixtures . Samples prepared twice from independent

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Representative electron micrographs of homofibrils formed by M451-90, RIPK1497‑583, His-Ub-RIPK3387‑518 or ZBP1170-355 individually, and heterofibrils formed by indicated mixtures.Samples prepared twice from independent protein preparations


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

Heterofibrils containing mCHERRY-M451-90 and YPet‑RIPK1497‑583 C Heterofibrils containing mCHERRY-M451-90 and YPet-RIPK3387‑518 D Heterofibrils containing mCHERRY-M451-90 and YPet‑ZBP1170‑355 E Heterofibrils containing mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑51


Heterofibrils containing mCHERRY - RIPK3387 ‑ 518 YPet ‑ ZBP1170 ‑ 355 Pearson ' s correlation coefficients measured from two confocal of indicated mixtures .

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Heterofibrils containing mCHERRY- RIPK3387‑518 and YPet‑ZBP1170‑355 Pearson's correlation coefficients measured from two confocal images of the indicated protein mixtures.


Heterofibrils containing ECFP-M451-90 with mCHERRY- RIPK1497‑583 and YPet-RIPK3387‑518


STED of heteromeric fibrils formed by co - assembly of mCHERRY - M451 - 90 YPet - RIPK3387 ‑

STED images of heteromeric fibrils formed by co-assembly of mCHERRY-M451-90 and YPet-RIPK3387‑518


SDS of monomeric or assembled forms of YPet - RIPK3387 ‑ 518 mCHERRY - M451 - 90WT or mCHERRY - M451 - 90AAAA . Monomeric forms of maintained in 8 M urea prior to electrophoresis , as indicated . Assembled samples incubated with 0 or 2 % SDS at RT for 10 min before electrophoresis . components as indicated above each lane .

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SDS AGE analysis of monomeric or assembled forms of YPet-RIPK3387‑518 and mCHERRY-M451-90WT or mCHERRY-M451-90AAAA. Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis, as indicated. Assembled samples incubated with 0 or 2% SDS at RT for 10 min before electrophoresis. Protein components as indicated above each lane.


SDS of assemblies of mCHERRY - RIPK1497 ‑ 583 YPet - RIPK3387 ‑ 518 with or without four times molar equivalents of His - Ub - M45 .

SDS AGE analysis of assemblies of mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 with or without four times molar equivalents of His-Ub-M45.


SDS of assemblies of YPet - RIPK3387 ‑ 518 mCHERRY - ZBP1170 - 355 with or without four times molar equivalents of His - Ub - M45 .

SDS AGE analysis of assemblies of YPet-RIPK3387‑518 and mCHERRY-ZBP1170-355 with or without four times molar equivalents of His-Ub-M45.


Confocal microscopy image of a single fibril composed of YPet - M451 -

Confocal microscopy image of a single fibril composed of YPet-M451-90


A , B BMDCs were treated with OxPAPC or DPPC ( 40 μg / ml ) for 60 min followed by R837 stimulation ( 5 μg / ml ) . ( A ) Supernatants were harvested after 18 h , concentrations of IL - 6 IL - 12 were quantified by ELISA . One - way ANOVA adjusted by Dunnett ' s multiple comparisons . Mean ± SD of triplicate determinations from > 3 independent experiments are shown .

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A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown.


A , B BMDCs were treated with OxPAPC or DPPC ( 40 μg / ml ) for 60 min followed by R837 stimulation ( 5 μg / ml ) . ( B ) mRNA was harvested after 2 h , expression of IL - 6 IL - 12 was measured by real - time PCR normalized to G6pdx . Unpaired two - t - . Data ( mean ± SD ) are representative of three independent experiments .

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A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.


C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC . Periods of oxidation were 24 h for CuSO4 ( 10 μM ) , 48 h for FeSO4 ( 10 μM ) , 72 h for air . BMDCs were treated for 60 min with lipids prior stimulation with R837 ( 5 μg / ml ) for 18 h . Cytokine concentrations in supernatants were quantified by ELISA . One - way ANOVA adjusted by Dunnett ' s multiple comparisons . Data represent mean ± of triplicate determinations .

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C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.


D PAPC ( 40 μg / ml ) oxidized with CuSO4 ( 10 μM ) for indicated times was used for treatment of BMDCs prior to R837 ( 5 μg / ml ) stimulation measurement of IL - 12 secretion . Mean ± of triplicate determinations from three different oxidation series are shown .

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

D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.


E , F Treatment of BMDCs with indicated concentrations of CuSO4 - oxidized PAPC suppressed IL - 6 ( E ) IL - 12 ( F ) secretion triggered by a variety of different TLR agonists . After OxPAPC treatment for 60 min , BMDCs were stimulated for 18 h with LTA ( 500 ng / ml ) , Poly I : C ( 50 μg / ml ) , LPS ( 10 ng / ml ) , R837 ( 5 μg / ml ) , CpG ( 100 nM ) . Cytokine concentrations in supernatant were quantified by ELISA . Data are shown as mean ± SD of triplicate determinations from three independent experiments were analyzed by one - way ANOVA adjusted by Dunnett ' s multiple comparisons .

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E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.


G - I Splenicdendritic were treated with OxPAPC or DPPC ( 40 μg / ml ) before co - culturing with naïve transgenic SMARTA CD4T in presence of specific peptide gp61 . ( G ) After 4 days of culture , T - polarization was assessed by intracellular for cytokines IL - 4 ( Th2 ) IFN - γ ( Th1 ) . ( H ) Bar graphs represent frequencies of IL - 4 - IFN - γ - producing T after 4 days of co - culture with OxPAPC - treated DPPC - treated splenicdendritic . Bars represent mean ± SD of duplicate experiments . * P ≤ 0 . 05 by unpaired two - t - . ( I ) IFN - γ production in supernatants of SMARTA CD4T ( stimulated with 1 , 000 nM gp61 ) co - cultured with OxPAPC - DPPC - treated splenicdendritic for 4 days . One - way ANOVA adjusted by Dunnett ' s multiple comparisons . Bars represent mean ± SD .

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G-I Splenicdendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenicdendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenicdendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.


Mass spectrometric of a variety of OxPL species obtained by CuSO4 - catalyzed oxidation of PAPC for 2 24 h . Mean ± of duplicate determinations are shown .

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

Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4-catalyzed oxidation of PAPC for 2 and 24 h. Mean ± SEM of duplicate determinations are shown.


Correlation between abundance of OxPL species detected in mixtures of differentially oxidized OxPL preparations capacity of respective overall OxPL mixtures to suppress IL - 12 secretion of thioglycollate - elicited macrophages . Dots represent data of OxPAPC preparations .

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

Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL-12 secretion of thioglycollate-elicited macrophages. Dots represent data of individual OxPAPC preparations.


Selected candidate lipids were tested for their inhibitory activity on R837 - induced ( 5 μg / ml ; 18 h ) cytokine secretion in BMDCs . Concentrations of indicated lipids : PECPC ( 10 μM ) , PEIPC ( 10 μM ) , OxPAPC ( 40 μg / ml ) , DPPC ( 40 μg / ml ) , POVPC ( 40 μM ) , PGPC ( 40 μM ) , KOdiAPC ( 40 μM ) . Representative data ( mean ± SD of triplicate determinations ) from one of three independent experiments are shown . * * * * P < 0 . 0001 ; ns , not significant ; as determined by one - way ANOVA adjusted by Sidak ' s multiple comparisons .

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Selected candidate lipids were tested for their inhibitory activity on R837-induced (5 μg/ml; 18 h) cytokine secretion in BMDCs. Concentrations of indicated lipids: PECPC (10 μM), PEIPC (10 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), and KOdiAPC (40 μM). Representative data (mean ± SD of triplicate determinations) from one of three independent experiments are shown. ****P < 0.0001; ns, not significant; as determined by one-way ANOVA adjusted by Sidak's multiple comparisons test.


Dose - response curves of IL - 12 secretion in BMDCs pulsed for 60 min with indicated lipids , followed by stimulation with R837 ( 5 μg / ml ) for 18 h . Toxic concentrations of lipids were excluded from . Mean ± of triplicate determinations are shown .

Dose-response curves of IL-12 secretion in BMDCs pulsed for 60 min with the indicated lipids, followed by stimulation with R837 (5 μg/ml) for 18 h. Toxic concentrations of lipids were excluded from analysis. Mean ± SEM of triplicate determinations are shown.


EC50 values of anti - inflammatory lipid products normalized to capacity of 15d - PGJ2 to suppress IL - 12 production in BMDCs ( left panel ) their respective bioactivities ( right panel ) depicted as fold increase relative to 15d - PGJ2 . Mean ± of triplicate determinations are shown .

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EC50 values of anti-inflammatory lipid products normalized to the capacity of 15d-PGJ2 to suppress IL-12 production in BMDCs (left panel) and their respective bioactivities (right panel) depicted as the fold increase relative to 15d-PGJ2. Mean ± SEM of triplicate determinations are shown.


IL - 12 production of BMDC stimulated via TLR 4 ( LPS ; 100 ng / ml ) , TLR 9 ( CpG ; 100 nM ) , TLR 7 ( R837 ; 5 μg / ml ) after pretreatment with indicated free esterified versions of EC 15d - PGJ2 . Lipids were used at 1 μM ( EC ) , 10 μM ( PECPC ) , or 20 μM ( 15d - PGJ2 15d - PGJ2PC ) . Data ( mean ± ) are representative of 3 independent experiments . * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ; * * * * P ≤ 0 . 0001 ; determined by one - way ANOVA adjusted by Dunnett ' s multiple comparisons .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-12 production of BMDC stimulated via TLR 4 (LPS; 100 ng/ml), TLR 9 (CpG; 100 nM), and TLR 7 (R837; 5 μg/ml) after pretreatment with the indicated free and esterified versions of EC and 15d-PGJ2. Lipids were used at 1 μM (EC), 10 μM (PECPC), or 20 μM (15d-PGJ2 and 15d-PGJ2PC). Data (mean ± SEM) are representative of 3 independent experiments. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; determined by one-way ANOVA adjusted by Dunnett's multiple comparisons test.


of capacity of various OxPL - derived species to license splenicdendritic to polarize naïve CD4T toward Th2 subset . Concentrations of indicated lipids : EC ( 1 μM ) , PECPC ( 10 μM ) , 15d - PGJ2 ( 20 μM ) , OxPAPC ( 40 μg / ml ) , DPPC ( 40 μg / ml ) , POVPC ( 40 μM ) , PGPC ( 40 μM ) , KOdiAPC ( 40 μM ) .

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Analysis of the capacity of various OxPL-derived species to license splenicdendritic cells to polarize naïve CD4T cells toward the Th2 subset. Concentrations of indicated lipids: EC (1 μM), PECPC (10 μM), 15d-PGJ2 (20 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), KOdiAPC (40 μM).


A IL - 12 production of BMDC from wild - type , Nrf2 − / − , Pparg − / − , Pparg litter mate control mice normalized to medium control ( open bars ) . were treated with indicated lipids ( filled bars ) for 60 min prior TLR 7 ligation with R837 ( 5 μg / ml ) for 18 h . Lipids were used at starting concentrations of 40 μM ( POVPC , PGPC , KOdiAPC ) , 40 μg / ml ( OxPAPC ) , 20 μM ( 15d - PGJ2 ) , 1 . 25 μM ( EC ) , depicted as black bars , 2 - fold serial dilutions thereof ( gray bars ) . Data represent mean ± of triplicates from one of three independent experiments .

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

A IL-12 production of BMDC from wild-type, Nrf2−/−, Pparg−/−, and Pparg litter mate control mice normalized to medium control (open bars). Cells were treated with the indicated lipids (filled bars) for 60 min prior TLR 7 ligation with R837 (5 μg/ml) for 18 h. Lipids were used at starting concentrations of 40 μM (POVPC, PGPC, and KOdiAPC), 40 μg/ml (OxPAPC), 20 μM (15d-PGJ2), and 1.25 μM (EC), depicted as black bars, and 2-fold serial dilutions thereof (gray bars). Data represent mean ± SEM of triplicates from one of three independent experiments.


B Expression of Nrf2 target genes Hmox1 Nqo1 in wild - type Nrf2 − / − BMDM stimulated with EC ( 2 μM ) for 60 min . Gene expression are presented relative to that of untreated after normalization to G6pdx . Data ( mean ± ) are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Expression of Nrf2 target genes Hmox1 and Nqo1 in wild-type and Nrf2−/−BMDM stimulated with EC (2 μM) for 60 min. Gene expression levels are presented relative to that of untreated cells after normalization to G6pdx. Data (mean ± SEM) are representative of two independent experiments.


C , D mRNA expression of Nrf2 targets Gclc Gsta3 ( C ) of pro - inflammatory cytokines IL - 6 IL - 12 ( D ) in wild - type , PPAR - γ - deficient , Nrf2 - deficient BMDM after treatment with EC or 15d - PGJ2 for 60 min followed by LPS treatment for 3 h . Expression are normalized to G6pdx . Data represent mean ± of triplicate cultures from one of two independent experiments .

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C, D mRNA expression levels of the Nrf2 targets Gclc and Gsta3 (C) and of the pro-inflammatory cytokines IL-6 and IL-12 (D) in wild-type, PPAR-γ-deficient, and Nrf2-deficient BMDM after treatment with EC or 15d-PGJ2 for 60 min followed by LPS treatment for 3 h. Expression levels are normalized to G6pdx. Data represent mean ± SEM of triplicate cultures from one of two independent experiments.


E mRNA expression of indicated chemokines as determined by qPCR . Wild - type BMDCs were treated with EC ( 1 μM ) or 15d - PGJ2 ( 20 μM ) for 60 min followed by R837 stimulation ( 5 μg / ml ) for 3 h . Expression are shown normalized to G6pdx . Data ( mean ± , n = 2 ) are representative of three independent experiments .

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E mRNA expression of the indicated chemokines as determined by qPCR. Wild-type BMDCs were treated with EC (1 μM) or 15d-PGJ2 (20 μM) for 60 min followed by R837 stimulation (5 μg/ml) for 3 h. Expression levels are shown normalized to G6pdx. Data (mean ± SEM, n = 2) are representative of three independent experiments.


A C57BL / 6mice were treated ( i . v . ) with 500 μg EC or DPPC control 2 h prior to i . p injection of 150 ng / g LPS together with 800 μg / g D - galactosamine . 4 h after LPS application , lungs were perfused with PBS embedded in paraffin . Tissue sections were hematoxylin - stained to visualize adherent . Bars represent 100 µm . B , C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image of lung tissue sections is presented for vessels in ( B ) as averages of single mice in ( C ) . Pooled data of two independent experiments are shown ( n = 10 for EC , n = 14 for DPPC ) . Unpaired two - t - .

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

A C57BL/6mice were treated (i.v.) with 500 μg EC or DPPC control 2 h prior to i.p injection of 150 ng/g LPS together with 800 μg/g D-galactosamine. 4 h after LPS application, lungs were perfused with PBS and embedded in paraffin. Tissue sections were hematoxylin-stained to visualize adherent cells. Bars represent 100 µm.B, C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in (B) and as averages of single mice in (C). Pooled data of two independent experiments are shown (n = 10 for EC, n = 14 for DPPC). Unpaired two-tailed t-test.


D , E C57BL / 6mice were treated with EC or DPPC by intra - tracheal instillation at 18 h ( 50 μg ) 1 . 5 h ( 100 μg ) prior to i . p . injection of 150 ng / g LPS 800 μg / g D - galactosamine . Bar graphs represent absolute numbers of total infiltrating of neutrophils ( D ) . Unpaired two - t - . Data represent mean ± from one of two independent experiments with at least 6 mice per group . ( E ) Dot plots depict exemplary gating of lungneutrophils on pregated CD45 + CD11c − SiglecF − BAL of EC - / LPS - treated DPPC - / LPS - treated mice .

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D, E C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils (D). Unpaired two-tailed t-test. Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.


F , G concentrations of IL - 6 ( F ) IL - 12 ( G ) in BAL of mice treated as in ( D ) were quantified by ELISA . Data ( mean ± ) are representative of two independent experiments with 3 mice per group . * * * * P < 0 . 0001 ; unpaired two - t - .

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F, G The concentrations of IL-6 (F) and IL-12 (G) in the BAL of mice treated as in (D) were quantified by ELISA. Data (mean ± SEM) are representative of two independent experiments with 3 mice per group. ****P < 0.0001; unpaired two-tailed t-test.


C57BL / 6mice were treated with EC or DPPC by intra - tracheal instillation at 18 h ( 50 μg ) 1 . 5 h ( 100 μg ) prior to i . p . injection of 150 ng / g LPS 800 μg / g D - galactosamine . ( E ) Dot plots depict exemplary gating of lungneutrophils on pregated CD45 + CD11c − SiglecF − BAL of EC - / LPS - treated DPPC - / LPS - treated mice .

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C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.


Dose - response curves showing modulation of R837 - induced ( 5 μg / ml ; 18 h ) IL - 12 secretion by prior treatment of BMDCs with indicated synthetic lipids for 1 h . Chemical structures ΔEC50 values of synthetic OxPL variant lipids analyzed in ( A ) , presented relative to EC .

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Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h.Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in (A), presented relative to EC.


of IL - 6 IL - 12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 ( 5 μg / ml ; 18 h ) . Bars represent mean ± from one of three independent experiments . * * P < 0 . 01 ; * * * P < 0 . 001 ; one - way ANOVA adjusted by Dunnett ' s multiple comparisons .

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Quantification of IL-6 and IL-12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 (5 μg/ml; 18 h). Bars represent mean ± SEM from one of three independent experiments. **P < 0.01; ***P < 0.001; one-way ANOVA adjusted by Dunnett's multiple comparisons test.


mRNA expression of Nrf2 targets Hmox1 Nqo1 normalized to G6pdx expression . BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation ( 5 μg / ml ) for 3 h . Data ( mean ± SD ) are representative of three independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - t - .

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mRNA expression of the Nrf2 targets Hmox1 and Nqo1 normalized to G6pdx expression. BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.


ΔEC50 values of indicated lipids OxPL shown relative to that of EC as determined in ( E ) . Dose - response curves showing modulation of R837 - induced ( 5 μg / ml ; 18 h ) IL - 12 secretion in BMDCs by prior treatment with indicated OxPL derivatives for 1 h .

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ΔEC50 values of the indicated lipids and OxPL shown relative to that of EC as determined in (E).Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion in BMDCs by prior treatment with the indicated OxPL derivatives for 1 h.


mRNA of indicated chemokines relative to G6pdx expression . BMDC were pretreated for 60 min with 500 nM cEC or variant BisRed prior to R837 stimulation ( 5 μg / ml ) for 3 h . Data ( mean ± SD ) are representative of three independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - t - .

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mRNA quantification of the indicated chemokines relative to G6pdx expression. BMDC were pretreated for 60 min with 500 nM cEC or the variant BisRed prior to R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.


characterization of infiltrates in BAL . Groups of six C57BL / 6mice were pretreated i . t . with 50 μg cEC , EC , or BisRed 24 2 h before challenge with 150 ng / g LPS in presence of 800 μg / g D - galactosamine . BAL was harvested 4 h after LPS injection , inflammatory were characterized by FACS . * * P < 0 . 01 ; * * * * P < 0 . 0001 ; one - way ANOVA with Sidak ' s multiple comparisons .

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Quantification and characterization of cellular infiltrates in BAL. Groups of six C57BL/6mice were pretreated i.t. with 50 μg cEC, EC, or BisRed 24 and 2 h before challenge with 150 ng/g LPS in the presence of 800 μg/g D-galactosamine. BAL was harvested 4 h after LPS injection, and inflammatory cells were characterized by FACS analysis. **P < 0.01; ****P < 0.0001; one-way ANOVA with Sidak's multiple comparisons test.


Comparison of capacity of cEC , EC , PECPC ( 1 μM ) to license splenicdendritic to polarize naïve CD4T into IFN - γ - producing ( Th1 ) IL - 4 - producing ( Th2 ) effector . Data ( mean ± SD ) are representative of two independent experiments . One - way ANOVA adjusted by Dunnett ' s multiple comparisons . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

Comparison of the capacity of cEC, EC, and PECPC (1 μM) to license splenicdendritic cells to polarize naïve CD4T cells into IFN-γ-producing (Th1) and IL-4-producing (Th2) effector cells. Data (mean ± SD) are representative of two independent experiments. One-way ANOVA adjusted by Dunnett's multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.


( C ) Representative confocal image of long propriospinal neurons expressing FGF22 ( magenta ) in cervical spinal cord .

(C) Representative confocal image of long propriospinal neurons expressing FGF22 (magenta) in cervical spinal cord.


( E ) Schematic illustration of hCST collateral into cervical cord confocal of hCST collaterals ( green ) , stained for vGlut ( red ) vGAT ( blue ) 21 days following spinal cord injury . Insets represent 3D views generated in Imaris of deconvolved confocal image . ( F ) of bouton density characterization of bouton between control FGF22 overexpressing mice ( n = 4 - 5 mice per group ) . (

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(E) Schematic illustration of the hCST collateral into the cervical cord and confocal images of hCST collaterals (green), stained for vGlut (red) and vGAT (blue) 21 days following spinal cord injury. Insets represent 3D views generated in Imaris of the deconvolved confocal image. (F) Quantification of bouton density and characterization of bouton between control and FGF22 overexpressing mice (n = 4-5 mice per group). (


( G ) Schematic illustration representative confocal image of LPSN viral labeling ( magenta ) CST collaterals ( green ) . Insets represent 3D views generated in Imaris of deconvolved confocal image . ( H ) of hCST contacts onto LPSNs characterization of vGlut onto LPSNs ( n = 4 - 5 mice per group ) .

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(G) Schematic illustration and representative confocal image of LPSN viral labeling (magenta) and CST collaterals (green). Insets represent 3D views generated in Imaris of the deconvolved confocal image. (H) Quantification of hCST contacts onto LPSNs and characterization of vGlut intensity onto individual LPSNs (n = 4-5 mice per group).


( C ) Schematic illustration representative confocal image for vGlut2 - Cre neuronal labeling ( cyan ) hCST collaterals ( ) . Insets represent 3D views generated in Imaris of confocal image . of hCST contacts onto vGlut neurons ( n = 4 - 6 mice per group ) .

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(C) Schematic illustration and representative confocal image for vGlut2-Cre neuronal labeling (cyan) and hCST collaterals (white). Insets represent 3D views generated in Imaris of the confocal image. Quantification of hCST contacts onto vGlut neurons (n = 4-6 mice per group).


( C ) Schematic illustration of hCST collateral exiting into cervical cord confocal showing exiting hCST collaterals in control ( left panel ) FGF22 treated ( right panel ) mice of number of hCST collaterals that enter cervical grey matter 3 following spinal cord injury . ( n = 6 mice per group ) .

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(C) Schematic illustration of the hCST collateral exiting into the cervical cord and confocal images showing exiting hCST collaterals in control (left panel) and FGF22 treated (right panel) mice and quantification of the number of hCST collaterals that enter the cervical grey matter 3 weeks following spinal cord injury. (n = 6 mice per group).


( E ) Schematic illustration of boutons on cervical hCST collateral confocal showing putative synaptic boutons ( arrows ) on newly formed cervical hCST collaterals at 3 following spinal cord injury in a FGF22 ( right ) a control ( left ) mice . of bouton density in control FGF22 treated mice . ( n = 6 mice per

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(E) Schematic illustration of the boutons on cervical hCST collateral and confocal images showing putative synaptic boutons (arrows) on newly formed cervical hCST collaterals at 3 weeks following spinal cord injury in a FGF22 (right) and a control (left) mice. Quantification of bouton density in control and FGF22 treated mice. (n = 6 mice per group


( F ) Schematic illustration of hCST contacts onto relay neurons in cervical cord confocal showing contacts between CST collaterals ( red ) cervical interneurons ( cyan ) . Insets represent 3D views generated in Imaris of confocal image . of contacts between hCST collaterals cervical interneurons neurons . ( n = 6 per group ) .

(F) Schematic illustration of the hCST contacts onto relay neurons in the cervical cord and confocal images showing contacts between CST collaterals (red) and cervical interneurons (cyan). Insets represent 3D views generated in Imaris of the confocal image. Quantification of the contacts between the hCST collaterals and cervical interneurons neurons. (n = 6 per group).


( G ) Confocal showing contacts between CST collaterals ( red ) long propriospinal neurons ( magenta ) transduced with FGF22 ( cyan ) . of contacts between hCST collaterals long propriospinal transduced with FGF22 or control ( DP refers to double positive ) . Insets on right are magnification of boxed on left . ( n = 6 mice per group ) .

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(G) Confocal images showing contacts between CST collaterals (red) and long propriospinal neurons (magenta) transduced with FGF22 (cyan). Quantification of the contacts between the hCST collaterals and long propriospinal transduced with FGF22 or control virus (DP refers to double positive cells). Insets on the right are magnification of the boxed area on the left. (n = 6 mice per group).


( C ) Timeline of experiment with FGF22 injection right after onset of spinal cord lesion ( top ) . of functional recovery in regular ( middle ) irregular ( bottom ) ladder rung in controls ( grey ) FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " ) , 14dpi 21dpi after spinal cord injury . ( n = 9 - 10 mice per group ) . ( D ) Timeline of experiment with FGF22 injection 24hrs after onset of spinal cord lesion ( top ) . of functional recovery in regular ( middle ) irregular ( bottom ) ladder rung in control ( grey ) FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " or 2 days " 2dpi for late treatment ) , 14dpi 21dpi after spinal cord injury . ( n = 9 - 10 per group ) . ( E ) Timeline of experiment with FGF22 injection 5 days after onset of spinal cord lesion ( top ) . of functional recovery in regular ( middle ) irregular ( bottom ) ladder rung in control ( grey ) FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " ) , 14dpi 21dpi after spinal cord injury . ( n = 9 - 10 per group ) . ) . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Timeline of the experiment with FGF22 injection right after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in controls (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi"), 14dpi and 21dpi after spinal cord injury. (n = 9-10 mice per group). (D) Timeline of the experiment with FGF22 injection 24hrs after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in control (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi" or 2 days "2dpi for the late treatment), 14dpi and 21dpi after spinal cord injury. (n = 9-10 per group). (E) Timeline of the experiment with FGF22 injection 5 days after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in control (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi"), 14dpi and 21dpi after spinal cord injury. (n = 9-10 per group).). (F


( F ) Representative confocal image of CST boutons ( green ) synapsin ( red ) of of bouton synapsin positive ( n = 6 - 7 mice per group ) . ( G ) Representative confocal of CST bouton ( green ) bassoon ( magenta ) of of boutons that are bassoon positive ( n = 5 - 6 mice per group ) .

(F) Representative confocal image of CST boutons (green) and synapsin staining (red) and quantification of the percentage of bouton synapsin positive (n = 6-7 mice per group). (G) Representative confocal images of CST bouton (green) and bassoon staining (magenta) and quantification of the percentage of boutons that are bassoon positive (n = 5-6 mice per group). D


( D ) Violin plots showing distribution of sheath polymerization speed for both modes of T6SS assemblies . 300 assemblies ( 100 from each biological replicate ; 3 biological replicates ) were analyzed over at least 3 min at 5 s intervals . central dash indicates median while upper lower dash lines indicate quartiles . Unpaired t - was performed ( ns : not significant ) .

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(D) Violin plots showing the distribution of sheath polymerization speed for both modes of T6SS assemblies. 300 assemblies (100 from each biological replicate; 3 biological replicates) were analyzed over at least 3 min at 5 s intervals. The central dash line indicates the median while the upper and lower dash lines indicate the quartiles. Unpaired t-test was performed (ns: not significant).


( D ) frequency of contact - dependent assembly ( left panel ) number of assemblies per per min ( right panel ) in from different strains as indicated on x - axis . Three biological replicates were performed for each . At least 500 were analyzed per replicate on 3D - SIM time - lapse imaging of sheath dynamics ( TssB - sfGFP ) in 3 min period . Bar graph ( left panel ) scatter dot plot ( right panel ) show mean ± SD . Unpaired t - was performed .

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(D) The frequency of contact-dependent assembly (left panel) and number of assemblies per cell per min (right panel) in cells from different strains as indicated on x-axis. Three biological replicates were performed for each strain. At least 500 cells were analyzed per replicate on 3D-SIM time-lapse imaging of sheath dynamics (TssB-sfGFP) in 3 min period. Bar graph (left panel) and scatter dot plot (right panel) show mean ± SD. Unpaired t-test was performed.


C . Western blot of CTRL # 1 or BRD9 - KO # 1 lysates from treated for indicated times with 1000 IU / mL of IFN - α2 . indicated were detected with specific antibodies . Data are representative of at least two biological replicates .

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C. Western blot analysis of CTRL#1 or BRD9-KO#1 lysates from cells treated for the indicated times with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates.


D . Immunofluorescence of CTRL # 1 or BRD9 - KO # 1 treated for indicated times with 1000 IU / mL of IFN - α2 . indicated were detected with specific antibodies . Data are representative of at least two biological replicates . Scale bars indicate 10μm .

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D. Immunofluorescence analysis of CTRL#1 or BRD9-KO#1 cells treated for the indicated times with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates. Scale bars indicate 10μm.


F . Western blot of CTRL # 1 or BRD9 - KO # 1 lysates from treated or not for 16h with 1000 IU / mL of IFN - α2 . indicated were detected with specific antibodies . Data are representative of at least two biological replicates .

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F. Western blot analysis of CTRL#1 or BRD9-KO#1 lysates from cells treated or not for 16h with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates.


I . CTRL # 1 or BRD9 - KO # 1 were stably - transduced with BRD9 - expressing , or control ( EV , empty vector ) , lentiviruses treated with 1000 IU / mL of IFN - α2 for 16h prior to infection with IAV ( WSN / 33 ) at an MOI of 0 . 01 PFU / . Viral titers were determined after 24h by plaque . Data information : data represent means standard deviations from n = 3 biological experiments ( data points shown ) . Statistical significance was determined by 1 - way ANOVA on log - transformed plaque ( * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 ) . Dotted lines are a visual guide for maximum minimum replication in control in absence presence of IFN - α2 , respectively . Numbers above IFN - α2 - treated bars indicate their approximate difference to respective untreated conditions .

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I. CTRL#1 or BRD9-KO#1 cells were stably-transduced with BRD9-expressing, or control (EV, empty vector), lentiviruses and treated with 1000 IU/mL of IFN-α2 for 16h prior to infection with IAV (WSN/33) at an MOI of 0.01 PFU/cell. Viral titers were determined after 24h by plaque assay. Data information: data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed plaque counts (**p-value < 0.001; ****p-value < 0.0001). Dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions.


D . A549 were treated with 125nM dBRD9 - A ( + ) or DMSO ( - ) for 6h prior stimulation with 1000 IU / mL of IFN - α2 for 16h . Following lysis , western blot was used to detect of indicated . Data are representative of at least two biological replicates .

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D. A549 cells were treated with 125nM dBRD9-A (+) or DMSO (-) for 6h prior stimulation with 1000 IU/mL of IFN-α2 for 16h. Following lysis, western blot was used to detect levels of the indicated proteins. Data are representative of at least two biological replicates.