( E ) RT - qPCR of relative miR - 21 - 3p level in epidermis of Ppard + / + - / - mice , acutely irradiated ( Ac - UV ; + ) or non - irradiated ( - ) , treated with PPAR / antagonist GSK0660 ( + ) or vehicle ( - ) , as indicated . miR - 21 - 3p : Ppard + / + no UV vs Ppard + / + Ac - UV P = 0 . 005 , Ppard + / + Ac - UV vs Ppard + / + Ac - UV / GSK0660 P = 0 . 04 , N = 5 ( Ppard + / + ) to 3 ( Ppard - / - ) per group . One representative experiment is shown out of two independent

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

(E) RT-qPCR quantification of relative miR-21-3p level in the epidermis of Ppard +/+ and -/-mice, acutely irradiated (Ac-UV; +) or non-irradiated (-), treated with the PPAR/ antagonist GSK0660 (+) or vehicle (-), as indicated. miR-21-3p: Ppard +/+ no UV vs Ppard +/+ Ac-UV P = 0.005, Ppard +/+ Ac-UV vs Ppard +/+ Ac-UV/GSK0660 P = 0.04, N=5 (Ppard +/+) to 3 (Ppard -/-) animals per group. One representative experiment is shown out of two independent replicates


( F ) RT - qPCR of relative miR - 21 - 3p in HaCat treated with PPAR / agonists GW501516 , GW0742 ( + ) or vehicle ( - ) as indicated . miR - 21 - 3p : Veh vs GW501516 P = 4E - 04 , Veh vs GW0742 P = 2E - 04 . N = 2 - 3 biological replicates , one representative experiment is shown out of two independent replicates .

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 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

(F) RT-qPCR quantification of relative miR-21-3p levels in HaCat cells treated with the PPAR/ agonists GW501516, GW0742 (+) or vehicle (-) as indicated. miR-21-3p: Veh vs GW501516 P = 4E-04, Veh vs GW0742 P = 2E-04. N=2-3 biological replicates, one representative experiment is shown out of two independent replicates.


( A ) RT - qPCR of relative pri - miR - 21 level in HaCaT human keratinocytes treated with PPAR / agonist GW0742 ( + ) or vehicle ( - ) , with ( + ) or without ( - ) cycloheximide ( Cyclo ) as indicated . Pri - miR - 21 : Veh vs GW0742 P = 0 . 009 . N = 3 biological replicates , one representative experiment is shown out of two independent replicates .

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

(A) RT-qPCR quantification of relative pri-miR-21 level in HaCaT human keratinocytes treated with the PPAR/ agonist GW0742 (+) or vehicle (-), with (+) or without (-) cycloheximide (Cyclo) as indicated. Pri-miR-21: Veh vs GW0742 P = 0.009. N=3 biological replicates, one representative experiment is shown out of two independent replicates.


( B ) RT - qPCR of relative pri - miR - 21 , pre - miR - 21 , miR - 21 - 5p miR - 21 - 3p in HaCaT treated for 24h with 2 or 5ng / ml of recombinant human TGFβ - 1 ( + ) or vehicle ( - ) as indicated . Pri - miR - 21 : Veh vs TGF - 1 5ng / ml P = 0 . 029 ; Pre - miR - 21 : Veh vs TGF - 1 5ng / ml P = 0 . 001 ; miR - 21 - 5p : Veh vs TGF - 1 5ng / ml P = 0 . 002 ; miR - 21 - 3p : Veh vs TGF - 1 5ng / ml P = 1 . 7E - 05 . N = 3 biological replicates , one representative experiment is shown out of two independent replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR quantification of relative pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in HaCaT cells treated for 24h with 2 or 5ng/ml of recombinant human TGFβ-1 (+) or vehicle (-) as indicated. Pri-miR-21: Veh vs TGF-1 5ng/ml P = 0.029; Pre-miR-21: Veh vs TGF-1 5ng/ml P = 0.001; miR-21-5p: Veh vs TGF-1 5ng/ml P = 0.002; miR-21-3p: Veh vs TGF-1 5ng/ml P = 1.7E-05. N=3 biological replicates, one representative experiment is shown out of two independent replicates .


( C ) RT - qPCR of pri - miR - 21 , pre - miR - 21 , miR - 21 - 5p miR - 21 - 3p in HaCat treated for 24h with PPAR / agonist GW0742 ( + ) , TGFβ receptor inhibitor SB431542 ( + ) , or vehicle ( - ) as indicated . Pri - miR - 21 : GW0742 vs SB431542 P = 0 . 004 , GW0742 vs GW0742 / SB431542 P = 0 . 015 ; Pre - miR - 21 : Veh vs GW0742 P = 0 . 022 , GW0742 vs SB431542 P = 0 . 036 , GW0742 vs GW0742 / SB431542 P = 0 . 024 ; miR - 21 - 5p : GW0742 vs SB431542 P = 0 . 034 , GW0742 vs GW0742 / SB431542 P = 0 . 024 ; miR - 21 - 3p : Veh vs GW0742 P = 0 . 036 , GW0742 vs SB431542 P = 0 . 011 , GW0742 vs GW0742 / SB431542 P = 0 . 008 . N = 3 biological replicates , one representative experiment is shown out of two independent replicates .

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

(C) RT-qPCR quantification of pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in HaCat cells treated for 24h with the PPAR/ agonist GW0742 (+), TGFβ receptor inhibitor SB431542 (+), or vehicle (-) as indicated. Pri-miR-21: GW0742 vs SB431542 P = 0.004, GW0742 vs GW0742/SB431542 P = 0.015; Pre-miR-21: Veh vs GW0742 P = 0.022, GW0742 vs SB431542 P = 0.036, GW0742 vs GW0742/SB431542 P = 0.024; miR-21-5p: GW0742 vs SB431542 P = 0.034, GW0742 vs GW0742/SB431542 P = 0.024; miR-21-3p: Veh vs GW0742 P = 0.036, GW0742 vs SB431542 P = 0.011, GW0742 vs GW0742/SB431542 P = 0.008. N=3 biological replicates, one representative experiment is shown out of two independent replicates.


( D ) RT - qPCR of relative Tgfb1 level in epidermis of acutely irradiated ( Ac - UV ) non - irradiated Ppard + / + - / - mice . Tgfb1 : Ppard + / + no UV vs Ac - UV P = 0 . 034 , Ppard + / + Ac - UV vs Ppard Ac - UV P = 0 . 030 . N = 2 - 3 per group , one representative experiment is shown out of three independent replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR quantification of relative Tgfb1 level in the epidermis of acutely irradiated (Ac-UV) and non-irradiated Ppard +/+ and -/- mice. Tgfb1: Ppard +/+ no UV vs Ac-UV P= 0.034, Ppard +/+ Ac-UV vs Ppard Ac-UV P= 0.030. N=2-3 animals per group, one representative experiment is shown out of three independent replicates.


( E ) RT - qPCR of relative miR - 21 - 5p miR - 21 - 3p in skin of Ppard + / + mice treated with TGFβ receptor inhibitor SB431542 ( + ) or vehicle ( - ) , with ( + ) or without ( - ) acute UV exposure ( Ac - UV ) . miR - 21 - 5p : no UV vs Ac - UV P = 0 . 038 , Ac UV vs Ac UV / SB431542 P = 0 . 028 ; miR - 21 - 3p : no UV vs Ac - UV P = 0 . 017 ; Ac - UV vs Ac - UV / SB431542 P = 0 . 035 . N = 3 per group , one representative experiment is shown out of three independent replicates .

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

(E) RT-qPCR quantification of relative miR-21-5p and miR-21-3p levels in the skin of Ppard +/+ mice treated with the TGFβ receptor inhibitor SB431542 (+) or vehicle (-), with (+) or without (-) acute UV exposure (Ac-UV). miR-21-5p: no UV vs Ac-UV P = 0.038, Ac UV vs Ac UV/SB431542 P = 0.028; miR-21-3p: no UV vs Ac-UV P = 0.017; Ac-UV vs Ac-UV/SB431542 P = 0.035. N=3 animals per group, one representative experiment is shown out of three independent replicates.


( A ) RT - qPCR of relative SMAD7 expression level in epidermis of acutely irradiated ( Ac - UV ; + ) ex vivo biopsies of human normal skin . SMAD7 : no UV vs Ac - UV P = 0 . 032 . N = 4 biological replicates , one representative experiment is shown out of three independent experiments performed with skin of three different donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR quantification of relative SMAD7 expression level in the epidermis of acutely irradiated (Ac-UV; +) ex vivo biopsies of human normal skin. SMAD7: no UV vs Ac-UV P = 0.032. N=4 biological replicates, one representative experiment is shown out of three independent experiments performed with the skin of three different donors.


( B ) Left panel : wild - type ( WT SMAD7 3 ' UTR ) mutated ( MUT SMAD7 3 ' UTR ; * : mutated nucleotides ) miR - 21 - 3p binding sequences in humanSMAD7 3 ' UTR . Right panel : Luciferase reporter with wild - type ( WT SMAD7 3 ' UTR ) or mutated ( MUT SMAD7 3 ' UTR ) SMAD7 3 ' UTR in HEK293 overexpressing miR - 21 - 3p ( miR - 21 - 3p mimic ) or a scrambled ( Control ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: wild-type (WT SMAD7 3'UTR) and mutated (MUT SMAD7 3'UTR; *: mutated nucleotides) miR-21-3p binding sequences in the humanSMAD7 3'UTR. Right panel: Luciferase reporter assay with wild-type (WT SMAD7 3'UTR) or mutated (MUT SMAD7 3'UTR) SMAD7 3'UTR in HEK293 cells overexpressing miR-21-3p (miR-21-3p mimic) or a scrambled sequence (Control).


( C ) Left panel : normalized expression data of SMAD7 mRNA obtained from genomic microarray of human HaCaT treated with a miR - 21 - 3p mimic ( miR - 21 - 3p mimic ) or a scrambled ( control ) . N = 3 biological replicates . Middle panel : western blot of SMAD7 level ( normalized to GAPDH level ) in human HaCaT treated with a miR - 21 - 3p mimic ( miR - 21 - 3p mimic ) or a scrambled ( control ) . SMAD7 : control vs miR - 21 - 3p mimic P = 0 . 031 . N = 3 biological replicates , Right panel : western blot of SMAD7 GAPDH in human HaCaT treated with a miR - 21 - 3p mimic ( miR - 21 - 3p mimic ) or a scrambled ( control ) , N = 3 biological replicates ; one representative experiment is shown out of two independent replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: normalized expression data of SMAD7 mRNA obtained from genomic microarray analysis of human HaCaT cells treated with a miR-21-3p mimic (miR-21-3p mimic) or a scrambled sequence (control). N=3 biological replicates. Middle panel: western blot quantification of SMAD7 protein level (normalized to GAPDH protein level) in human HaCaT cells treated with a miR-21-3p mimic (miR-21-3p mimic) or a scrambled sequence (control). SMAD7: control vs miR-21-3p mimic P = 0.031. N=3 biological replicates, Right panel: western blot of SMAD7 and GAPDH proteins in human HaCaT cells treated with a miR-21-3p mimic (miR-21-3p mimic) or a scrambled sequence (control), N=3 biological replicates; one representative experiment is shown out of two independent replicates.


( D ) Western blot of Smad7 from epidermis of acutely irradiated ( Ac - UV ; + ) Ppard + / + - / - mice . Loading control : GAPDH .

O O O O O O O O O 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) Western blot of Smad7 from epidermis of acutely irradiated (Ac-UV; +) Ppard +/+ and -/- mice. Loading control: GAPDH.


( E ) RT - qPCR of relative TGFB1 , SERPINE1 , p21 , RUNX SNAI2 in human HaCaT treated with a miR - 21 - 3p mimic ( miR - 21 - 3p mimic ) or a scrambled ( control ) with ( + ) or without ( - ) treatment with 2ng / ml recombinant TGFβ - 1 . SERPINE1 : TGF - 1 2ng / ml control vs miR - 21 - 3p mimic P = 0 . 004 ; p21 : TGF - 1 2ng / ml control vs miR - 21 - 3p mimic P = 0 . 03 ; RUNX : TGF - 1 2ng / ml control vs miR - 21 - 3p mimic P = 0 . 04 . N = 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR quantification of relative TGFB1, SERPINE1, p21, RUNX and SNAI2 levels in human HaCaT cells treated with a miR-21-3p mimic (miR-21-3p mimic) or a scrambled sequence (control) with (+) or without (-) treatment with 2ng/ml recombinant TGFβ-1. SERPINE1: TGF-1 2ng/ml control vs miR-21-3p mimic P = 0.004; p21: TGF-1 2ng/ml control vs miR-21-3p mimic P =0.03; RUNX: TGF-1 2ng/ml control vs miR-21-3p mimic P = 0.04. N=3 biological replicates.


( C ) RT - qPCR of IL6 , IL1B , IL1RAP , PTGS2 , CCL5 , CXCL10 CASP14 in HaCat transfected with a miR - 21 - 3p mimic or a scrambled ( control ) . IL6 : control vs miR - 21 - 3p mimic P = 0 . 05 ; IL1B : control vs miR - 21 - 3p mimic P = 0 . 005 ; IL1RAP : control vs miR - 21 - 3p mimic P = 0 . 008 ; PTGS2 : control vs miR - 21 - 3p mimic P = 0 . 050 ; CCL5 : control vs miR - 21 - 3p mimic P = 0 . 001 ; CXCL10 : control vs miR - 21 - 3p mimic P = 0 . 012 ; CASP14 : control vs miR - 21 - 3p mimic P = 9E - 05 . N = 3 biological replicates , one representative experiment is shown out of three independent replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR analysis of IL6, IL1B, IL1RAP, PTGS2, CCL5, CXCL10 and CASP14 levels in HaCat cells transfected with a miR-21-3p mimic or a scrambled sequence (control). IL6: control vs miR-21-3p mimic P = 0.05; IL1B: control vs miR-21-3p mimic P = 0.005; IL1RAP: control vs miR-21-3p mimic P = 0.008; PTGS2: control vs miR-21-3p mimic P = 0.050; CCL5: control vs miR-21-3p mimic P = 0.001; CXCL10: control vs miR-21-3p mimic P = 0.012; CASP14: control vs miR-21-3p mimic P = 9E-05. N=3 biological replicates, one representative experiment is shown out of three independent replicates.


( A ) RT - qPCR of Il6 , Il1b , Ptgs2 Ccl5 in epidermis of acutely irradiated ( Ac - UV ; + ) non - irradiated ( - ) Ppard + / + - / - mice . Il6 : Ppard + / + no UV vs Ac - UV P = 7E - 04 , Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 008 ; Il1b : Ppard + / + no UV vs Ac - UV P = 0 . 010 , Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 016 , Ppard - / - no UV vs Ac - UV P = 0 . 015 ; Ptgs2 : Ppard + / + no UV vs Ac - UV P = 0 . 028 , Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 028 , Ppard - / - no UV vs Ac - UV P = 8E - 04 ; Ccl5 : Ppard + / + no UV vs Ac - UV P = 0 . 002 , Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 014 , Ppard - / - no UV vs Ac - UV P = 0 . 010 ; ns : non significant . N = 4 per group , one representative experiment is shown out of three independent replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR quantification of Il6, Il1b, Ptgs2 and Ccl5 levels in the epidermis of acutely irradiated (Ac-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. Il6: Ppard +/+ no UV vs Ac-UV P = 7E-04, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.008; Il1b: Ppard +/+ no UV vs Ac-UV P = 0.010, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.016, Ppard -/- no UV vs Ac-UV P = 0.015; Ptgs2: Ppard +/+ no UV vs Ac-UV P = 0.028, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.028, Ppard -/- no UV vs Ac-UV P = 8E-04; Ccl5: Ppard +/+ no UV vs Ac-UV P = 0.002, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.014, Ppard -/- no UV vs Ac-UV P = 0.010; ns: non significant. N=4 animals per group, one representative experiment is shown out of three independent replicates.


( B ) Left panel : Immunostaining for infiltrating F4 / 80 - positive macrophages in acutely irradiated ( Ac - UV ) non - irradiated ( no UV ) Ppard + / + - / - miceskin sections . Magnification bar : 200m . Right panel : RT - qPCR of relative of macrophage marker Emr1 mRNA in total skin of acutely irradiated ( Ac - UV ; + ) non - irradiated ( - ) Ppard + / + - / - mice . Emr : Ppard + / + no UV vs Ac UV P = 0 . 034 ; Ppard + / + Ac UV vs Ppard - / - Ac UV P = 0 . 033 . N = 4 per group , one representative experiment is shown out of three independent replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: Immunostaining for infiltrating F4/80-positive macrophages in acutely irradiated (Ac-UV) and non-irradiated (no UV) Ppard +/+ and -/- miceskin sections. Magnification bar: 200m. Right panel: RT-qPCR quantification of relative levels of the macrophage marker Emr1 mRNA in total skin of acutely irradiated (Ac-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. Emr: Ppard +/+ no UV vs Ac UV P = 0.034; Ppard +/+ Ac UV vs Ppard -/- Ac UV P = 0.033. N=4 animals per group, one representative experiment is shown out of three independent replicates.


( C ) RT - qPCR of relative of Il6 , Il1b , Ptgs2 Tnfa in mouseepidermis acutely irradiated ( Ac - UV ; + ) non - irradiated ( - ) , treated with miR - 21 - 3p inhibitor ( + ) or mismatched control ( - ) as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR quantification of relative levels of Il6, Il1b, Ptgs2 and Tnfa in mouseepidermis acutely irradiated (Ac-UV; +) and non-irradiated (-), treated with miR-21-3p inhibitor (+) or mismatched control (-) as indicated.


( A ) RT - qPCR of relative of pri - miR - 21 , pre - miR - 21 , miR - 21 - 5p miR - 21 - 3p in healthy human skin human squamous carcinomas ( SCC ) . pri - miR - 21 Healthy skin vs SCC P = 0 . 043 ; pre - miR - 21 Healthy skin vs SCC P = 3E - 05 ; miR - 21 - 5p Healthy skin vs SCC P = 7E - 05 ; miR - 21 - 3p Healthy skin vs SCC P = 1 . 4E - 06 . ≥ 5 independent biopsies per 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR quantification of relative levels of pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in healthy human skin and human squamous cell carcinomas (SCC). pri-miR-21 Healthy skin vs SCC P = 0.043; pre-miR-21 Healthy skin vs SCC P = 3E-05; miR-21-5p Healthy skin vs SCC P = 7E-05; miR-21-3p Healthy skin vs SCC P = 1.4E-06. ≥ 5 independent biopsies per condition.


( B ) RT - qPCR of relative of pri - miR - 21 , pre - miR - 21 , miR - 21 - 5p miR - 21 - 3p in healthy human skin humanpsoriasis lesions . pri - miR - 21 Healthy skin vs Psoriasis P = 0 . 034 ; miR - 21 - 5p Healthy skin vs Psoriasis P = 0 . 034 ; miR - 21 - 3p Healthy skin vs Psoriasis P = 0 . 036 ; ns : non significant , ≥ 4 independent biopsies per 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 O O O O O O O O O O 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) RT-qPCR quantification of relative levels of pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in healthy human skin and humanpsoriasis lesions. pri-miR-21 Healthy skin vs Psoriasis P = 0.034; miR-21-5p Healthy skin vs Psoriasis P = 0.034; miR-21-3p Healthy skin vs Psoriasis P = 0.036; ns: non significant, ≥ 4 independent biopsies per condition.


( C ) RT - qPCR of relative of IL6 , IL1B , PTGS2 TNFA in epidermis of abdominal healthy humanskin biopsies exposed to acute UV ex vivo , with or without topical treatment with miR - 21 - 3p inhibitor ( + ) or mismatched control ( - ) as indicated . ns : not significant . IL6 : no UV vs Ac - UV P = 0 . 036 , Ac - UV vs Ac - UV / miR - 21 - 3p inhibitor P = 0 . 019 ; IL1B : no UV vs Ac - UV P = 0 . 002 , Ac - UV vs Ac - UV / miR - 21 - 3p inhibitor P = 0 . 023 ; PTGS2 : no UV vs Ac - UV P = 0 . 008 , Ac - UV vs Ac - UV / miR - 21 - 3p inhibitor P = 0 . 022 ; TNFA : no UV vs Ac - UV P = 0 . 032 , ns = non significant . ≥ 7 replicates , a pool of three independent experiments performed with skin of three different donors is 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR quantification of relative levels of IL6, IL1B, PTGS2 and TNFA levels in epidermis of abdominal healthy humanskin biopsies exposed to acute UV ex vivo, with or without topical treatment with miR-21-3p inhibitor (+) or mismatched control (-) as indicated. ns: not significant. IL6: no UV vs Ac-UV P = 0.036, Ac-UV vs Ac-UV/miR-21-3p inhibitor P = 0.019; IL1B: no UV vs Ac-UV P = 0.002, Ac-UV vs Ac-UV/miR-21-3p inhibitor P = 0.023; PTGS2: no UV vs Ac-UV P = 0.008, Ac-UV vs Ac-UV/miR-21-3p inhibitor P = 0.022; TNFA: no UV vs Ac-UV P = 0.032, ns = non significant. ≥ 7 replicates, a pool of three independent experiments performed with the skin of three different donors is shown.


A COS ‐ 1 co ‐ transfected with GFP ‐ ATP8A1 myc ‐ CDC50A were stained for myc ‐ tag TfnR , TGN46 , GM130 , VPS26 , CD63 , or LAMP2 . mCherry ‐ Rab11 was expressed in a in top row . Nuclei were stained with DAPI ( in blue ) . Pearson ' s coefficient between GFP ‐ ATP8A1 each organelle marker is shown in merged image ( mean ± SD , n > 20 from two experiments ) . Scale bars , 10 μm .

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A COS‐1 cells co‐transfected with GFP‐ATP8A1 and myc‐CDC50A were stained for myc‐tag and TfnR, TGN46, GM130, VPS26, CD63, or LAMP2. mCherry‐Rab11 was expressed in a cell in the top row. Nuclei were stained with DAPI (in blue). Pearson's coefficient between GFP‐ATP8A1 and each organelle marker is shown in the merged image (mean ± SD, n > 20 cells from two experiments). Scale bars, 10 μm.


B COS ‐ 1 were treated with ATP8A1 siRNAs for 72 h . lysates were prepared then immunoblotted with anti ‐ ATP8A1 antibody . α ‐ tubulin was used as a loading control .

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B COS‐1 cells were treated with ATP8A1 siRNAs for 72 h. Cell lysates were prepared and then immunoblotted with anti‐ATP8A1 antibody. α‐tubulin was used as a loading control.


C , D COS ‐ 1 were treated with ATP8A1 siRNA1 for 72 h . siRNA ‐ resistant GFP ‐ ATP8A1 ( WT or E191Q ) myc ‐ CDC50A were transfected 48 h after siRNA transfection . were pulsed with Alexa 594 ‐ Tfn chased in medium with unlabeled Tfn . During chase , were imaged every 6 min for 54 min . of Alexa 594 ‐ Tfn is shown as of that at 0 min ( mean ± , n = 4 - 6 ; * * * P 0 . 001 ; Student ' s t ‐ ) . See Supplementary Fig S2 for representative .

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C, D COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h. siRNA‐resistant GFP‐ATP8A1 (WT or E191Q) and myc‐CDC50A were transfected 48 h after siRNA transfection. Cells were pulsed with Alexa 594‐Tfn and chased in medium with unlabeled Tfn. During the chase, cells were imaged every 6 min for 54 min. The fluorescence intensity of Alexa 594‐Tfn is shown as percentage of that at 0 min (mean ± SEM, n = 4-6; ***P 0.001; Student's t‐test). See Supplementary Fig S2 for the representative images.


E , F COS ‐ 1 were treated with ATP8A1 siRNA1 for 72 h . were pulsed with Alexa 594 ‐ CTxB for 5 min chased for 15 or 60 min . were then fixed stained for GM130 . In ( E ) , representative are shown . In ( F ) , Pearson ' s coefficient between CTxB GM130 after 60 ‐ min chase is shown . Data represent mean ± SD ( n > 26 from three independent experiments ) . * * * P 0 . 001 ; two ‐ Student ' s t ‐ . Scale bars , 10 μm .

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E, F COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h. Cells were pulsed with Alexa 594‐CTxB for 5 min and chased for 15 or 60 min. Cells were then fixed and stained for GM130. In (E), representative images are shown. In (F), the Pearson's coefficient between CTxB and GM130 after 60‐min chase is shown. Data represent mean ± SD (n > 26 cells from three independent experiments). ***P 0.001; two‐tailed Student's t‐test. Scale bars, 10 μm.


A COS ‐ 1 were treated with ATP8A1 siRNA1 for 72 h stained for TfnR . Each image represents maximum z ‐ projection of four confocal slices . Intensified are shown in bottom row . Red dashed lines arrowheads indicate nuclei TfnR , respectively . Scale bars , 10 μm .

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A COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h and stained for TfnR. Each image represents the maximum intensity z‐projection of four confocal slices. Intensified images are shown in the bottom row. Red dashed lines and arrowheads indicate nuclei and TfnR puncta, respectively. Scale bars, 10 μm.


B COS ‐ 1 were treated with ATP8A1 siRNA1 for 72 h stained for indicated . Scale bars , 10 μm .

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B COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h and stained for the indicated proteins. Scale bars, 10 μm.


C COS ‐ 1 were treated with ATP8A1 siRNA1 for 72 h . TfnR ‐ GFP was transfected 48 h after siRNA transfection . were then imaged with an epifluorescent microscope . Selected stills from Supplementary Movie S1 are shown . asterisks indicate tips of tubules of interest . Scale bars , 5 μm .

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C COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h. TfnR‐GFP was transfected 48 h after siRNA transfection. Cells were then imaged with an epifluorescent microscope. Selected stills from Supplementary Movie S1 are shown. The asterisks indicate the tips of the tubules of interest. Scale bars, 5 μm.


D effect of increasing of 1 , 2 ‐ dioleoyl ‐ PS on ATPase activity of purified ATP8A1 ( WT E191Q ) ‐ CDC50A . Km Vmax for WT were 59 . 1 ± 3 . 5 μM 38 . 7 ± 0 . 8 μmol ATP / min / mg , respectively .

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D The effect of increasing levels of 1,2‐dioleoyl‐PS on the ATPase activity of purified ATP8A1 (WT and E191Q)‐CDC50A complex. The Km and Vmax for the WT complex were 59.1 ± 3.5 μM and 38.7 ± 0.8 μmol ATP/min/mg protein, respectively.


E COS ‐ 1 were treated with ATP8A1 siRNA1 for 72 h . siRNA ‐ resistant ATP8A1 ( WT or E191Q ) , myc ‐ CDC50A were transfected 48 h after siRNA transfection . were then fixed stained for TfnR . graph shows percentages of showing aberrant TfnR ‐ positive tubules as mean ± SD of three independent experiments . At least 50 were counted per experiment .

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E COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h. siRNA‐resistant ATP8A1 (WT or E191Q), and myc‐CDC50A were transfected 48 h after siRNA transfection. Cells were then fixed and stained for TfnR. The graph shows percentages of cells showing aberrant TfnR‐positive tubules as mean ± SD of three independent experiments. At least 50 cells were counted per experiment.


A COS ‐ 1 were treated with EHD1 siRNAs for 72 h . lysates were prepared then immunoblotted with EHD1 antibody . α ‐ tubulin was used as a loading control .

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A COS‐1 cells were treated with EHD1 siRNAs for 72 h. Cell lysates were prepared and then immunoblotted with EHD1 antibody. α‐tubulin was used as a loading control.


B , C were treated with EHD1 siRNA1 for 72 h . were pulsed with Alexa 488 ‐ Tfn chased in medium with unlabeled Tfn . During chase , were imaged every 3 min for 57 min . In ( B ) , representative of Alexa 488 ‐ Tfn in control or EHD1 ‐ depleted are shown . In ( C ) , of Alexa 488 ‐ Tfn is shown as of that at 0 min ( mean ± , n > 3 from three independent experiments ) . * * * P 0 . 001 ; two ‐ Student ' s t ‐ . Scale bars , 10 μm .

B, C Cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 488‐Tfn and chased in medium with unlabeled Tfn. During the chase, cells were imaged every 3 min for 57 min. In (B), representative images of Alexa 488‐Tfn in control or EHD1‐depleted cells are shown. In (C), the fluorescence intensity of Alexa 488‐Tfn is shown as percentage of that at 0 min (mean ± SEM, n > 3 cells from three independent experiments). ***P 0.001; two‐tailed Student's t‐test. Scale bars, 10 μm.


D COS ‐ 1 were treated with EHD1 siRNA1 for 72 h . were pulsed with Alexa 594 ‐ CTxB for 5 min chased for 60 min . were then fixed stained for GM130 . Pearson ' s coefficient between CTxB GM130 is shown ( mean ± SD , n > 30 from two independent experiments ) . * * * P 0 . 001 ; two ‐ Student ' s t ‐ . See Supplementary Fig S7A for representative .

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D COS‐1 cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 594‐CTxB for 5 min and chased for 60 min. Cells were then fixed and stained for GM130. The Pearson's coefficient between CTxB and GM130 is shown (mean ± SD, n > 30 cells from two independent experiments). ***P 0.001; two‐tailed Student's t‐test. See Supplementary Fig S7A for the representative images.


E COS ‐ 1 were treated with EHD1 siRNA1 for 72 h stained for TfnR . image represents maximum z ‐ projection of four confocal slices . Scale bar , 10 μm .

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E COS‐1 cells were treated with EHD1 siRNA1 for 72 h and stained for TfnR. The image represents the maximum intensity z‐projection of four confocal slices. Scale bar, 10 μm.


F COS ‐ 1 co ‐ transfected with GFP ‐ ATP8A1 myc ‐ CDC50A were stained for EHD1 . Scale bar , 10 μm .

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F COS‐1 cells co‐transfected with GFP‐ATP8A1 and myc‐CDC50A were stained for EHD1. Scale bar, 10 μm.


G were treated with ATP8A1 siRNA1 for 72 h stained for EHD1 . For rescue experiments , siRNA ‐ resistant GFP ‐ ATP8A1 ( WT or E191Q ) myc ‐ CDC50A were transfected 48 h after siRNA transfection . Asterisks indicate with GFP ‐ ATP8A1 expression . graph represents of with perinuclear localization of EHD1 as mean ± SD of three independent experiments ( at least 141 were counted for each condition ) . Scale bars , 10 μm .

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G Cells were treated with ATP8A1 siRNA1 for 72 h and stained for EHD1. For the rescue experiments, siRNA‐resistant GFP‐ATP8A1 (WT or E191Q) and myc‐CDC50A were transfected 48 h after siRNA transfection. Asterisks indicate cells with GFP‐ATP8A1 expression. The graph represents percentage of cells with perinuclear localization of EHD1 as mean ± SD of three independent experiments (at least 141 cells were counted for each condition). Scale bars, 10 μm.


A effect of various lipids on ATPase activity of purified ATP8A1 ‐ CDC50A . ATPase activity was measured in presence of 5 mM ATP 10 mol % of one of following lipids : 1 , 2 ‐ dioleoyl ‐ phosphatidycholine ( PC ) , 1 , 2 ‐ dioleoyl ‐ PS , 1 , 2 ‐ dioleoyl ‐ PE , 1 , 2 ‐ dioleoyl ‐ phosphatidylglycerol ( PG ) , 1 , 2 ‐ dioleoyl ‐ phosphatidylinositol ( PI ) , 1 , 2 ‐ dioleoyl ‐ phosphatidic acid ( PA ) , sphingomyelin ( SM ) , cholesterol ( Chol ) , or brain polar lipid ( BPL ) 90 % PC . activity of ATP8A1 ( E191Q ) ‐ CDC50A in presence of 1 , 2 ‐ dioleoyl ‐ PS was also determined .

A The effect of various lipids on the ATPase activity of the purified ATP8A1‐CDC50A complex. The ATPase activity was measured in the presence of 5 mM ATP and 10 mol % of one of the following lipids: 1,2‐dioleoyl‐phosphatidycholine (PC), 1,2‐dioleoyl‐PS, 1,2‐dioleoyl‐PE, 1,2‐dioleoyl‐phosphatidylglycerol (PG), 1,2‐dioleoyl‐phosphatidylinositol (PI), 1,2‐dioleoyl‐phosphatidic acid (PA), sphingomyelin (SM), cholesterol (Chol), or brain polar lipid (BPL) and 90% PC. The activity of the ATP8A1 (E191Q)‐CDC50A in the presence of 1,2‐dioleoyl‐PS was also determined.


B NBD ‐ labeled PS or PE transport ( flippase ) activity of ATP8A1 ( WT E191Q ) ‐ CDC50A . of NBD ‐ lipid flipped was shown .

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B NBD‐labeled PS or PE transport (flippase) activity of ATP8A1 (WT and E191Q)‐CDC50A complex. The percentage of NBD‐lipid flipped was shown.


D , E PSA ‐ 3 R ‐ 41 were transiently transfected with GFP ‐ EHD1 then cultured under indicated conditions for 72 h . were then fixed , nuclei were strained with DAPI . Arrowheads indicate perinuclear RE localization of GFP ‐ EHD1 . Scale bars , 10 μm .

D, E PSA‐3 and R‐41 cells were transiently transfected with GFP‐EHD1 and then cultured under the indicated conditions for 72 h. Cells were then fixed, and the nuclei were strained with DAPI. Arrowheads indicate the perinuclear RE localization of GFP‐EHD1. Scale bars, 10 μm.


A His ‐ tagged EHD1 was mixed with PC ‐ liposomes containing indicated % ( mol / mol ) of PS or PE . After 15 min , mixture was spun at 100 , 000 g for 30 min , resultant supernatant ( S ) pellet ( P ) were subjected to SDS - PAGE . lipids were then stained with Coomassie blue . B , C intensities of bands in ( A ) were quantified with ImageJ , of bound was calculated . In ( B ) , data represent mean ± SD of n = 3 independent experiments were analyzed with one ‐ way of variance ( ANOVA ) followed by Tukey - Kramer post hoc . * P 0 . 01 ; NS , not significant . In ( C ) , each point represents mean of two independent experiments with an error bar showing range of values obtained . sigmoidal fitting was applied .

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A His‐tagged EHD1 was mixed with PC‐based liposomes containing the indicated % (mol/mol) of PS or PE. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The proteins and lipids were then stained with Coomassie blue.B, C The intensities of individual bands in (A) were quantified with ImageJ, and the percentage of bound proteins was calculated. In (B), the data represent the mean ± SD of n = 3 independent experiments and were analyzed with one‐way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test. *P 0.01; NS, not significant. In (C), each point represents the mean of two independent experiments with an error bar showing the range of values obtained. The sigmoidal fitting was applied.


D , E His ‐ tagged EHD1 was mixed with PC ‐ liposomes ( 20 , 40 , or 60 % PS ) of decreasing produced by extrusion through filter pores of indicated . After 15 min , mixture was spun at 100 , 000 g for 30 min , resultant supernatant ( S ) pellet ( P ) were subjected to SDS - PAGE . lipids were then stained with Coomassie blue . In ( E ) , intensities of bands were quantified ( mean ± of three independent experiments ) . See Supplementary Fig S8 for distributions of prepared liposomes .

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D, E His‐tagged EHD1 was mixed with PC‐based liposomes (20, 40, or 60% PS) of decreasing size produced by extrusion through filter pores of the size indicated. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The proteins and lipids were then stained with Coomassie blue. In (E), the intensities of individual bands were quantified (mean ± SEM of three independent experiments). See Supplementary Fig S8 for the size distributions of the prepared liposomes.


A His ‐ tagged 2xPH was mixed with liposomes that were composed of 75 % ( mol / mol ) PE , 20 % ( mol / mol ) PC , 5 % ( mol / mol ) of PS , PA , PI , PIPs , or sulfatide . In case of PE / PC , liposomes of 80 % ( mol / mol ) PE 20 % ( mol / mol ) PC were used . After 15 min , mixture was spun at 100 , 000 g for 30 min , resultant supernatant ( S ) pellet ( P ) were subjected to SDS - PAGE . gels were stained with Coomassie blue .

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A His‐tagged 2xPH was mixed with liposomes that were composed of 75% (mol/mol) PE, 20% (mol/mol) PC, and 5% (mol/mol) of PS, PA, PI, PIPs, or sulfatide. In the case of PE/PC, liposomes of 80% (mol/mol) PE and 20% (mol/mol) PC were used. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The gels were stained with Coomassie blue.


B COS ‐ 1 were incubated in medium containing Alexa 488 ‐ Tfn for 40 min , washed , fixed , stained with recombinant 2xPH . Magnified image is shown in right panel . Scale bar , 10 μm .

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B COS‐1 cells were incubated in medium containing Alexa 488‐Tfn for 40 min, washed, fixed, and stained with recombinant 2xPH. Magnified image is shown in the right panel. Scale bar, 10 μm.


C COS ‐ 1 were transiently transfected with GFP ‐ lact ‐ C2 stained for Rab11 . Scale bar , 10 μm .

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C COS‐1 cells were transiently transfected with GFP‐lact‐C2 and stained for Rab11. Scale bar, 10 μm.


D , E COS ‐ 1 were transiently transfected with GFP ‐ lact ‐ C2 stained with recombinant 2xPH anti ‐ GM130 antibody . asterisk indicates a expressing GFP ‐ lact ‐ C2 , arrowhead indicates a non ‐ expressing . graph in ( E ) shows per pixel of recombinant 2xPH stain in RE , which was delimited by GM130 . Data are normalized to 2xPH in GFP ‐ lact ‐ C2 ( − ) ( mean ± SD , n = 13 for each condition ) . Scale bar , 10 μm .

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D, E COS‐1 cells were transiently transfected with GFP‐lact‐C2 and stained with recombinant 2xPH and anti‐GM130 antibody. The asterisk indicates a cell expressing GFP‐lact‐C2, and the arrowhead indicates a non‐expressing cell. The graph in (E) shows fluorescence intensity per pixel of recombinant 2xPH stain in RE area, which was delimited by GM130. Data are normalized to the 2xPH intensity in GFP‐lact‐C2 (−) cells (mean ± SD, n = 13 cells for each condition). Scale bar, 10 μm.


F - H COS ‐ 1 stably expressing GFP ‐ lact ‐ C2 were treated with ATP8A1 siRNA1 or EHD1 siRNA1 for 72 h stained with recombinant 2xPH anti ‐ GM130 antibody . intensities of 2xPH GFP ‐ lact ‐ C2 in RE were measured . of 2xPH ( G ) of 2xPH to GFP ‐ lact ‐ C2 intensities ( H ) are shown . In ( H ) , data are normalized to that of control . Data represent mean ± SD were analyzed with two ‐ Student ' s t ‐ ( n > 26 from three independent experiments ) . * * P 0 . 01 ; * * * P 0 . 001 ; NS , not significant . Scale bars , 10 μm .

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F-H COS‐1 cells stably expressing GFP‐lact‐C2 were treated with ATP8A1 siRNA1 or EHD1 siRNA1 for 72 h and stained with recombinant 2xPH and anti‐GM130 antibody. The intensities of 2xPH staining and GFP‐lact‐C2 in RE area were measured. The intensity of 2xPH (G) and the ratio of 2xPH to GFP‐lact‐C2 intensities (H) are shown. In (H), data are normalized to that of control cells. Data represent mean ± SD and were analyzed with two‐tailed Student's t‐test (n > 26 cells from three independent experiments). **P 0.01; ***P 0.001; NS, not significant. Scale bars, 10 μm.


A COS ‐ 1 co ‐ transfected with GFP ‐ ATP8A2 ( WT ) myc ‐ CDC50A were stained for myc ‐ tag TfnR . Scale bar , 10 μm .

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A COS‐1 cells co‐transfected with GFP‐ATP8A2 (WT) and myc‐CDC50A were stained for myc‐tag and TfnR. Scale bar, 10 μm.


B effect of increasing of PS on ATPase activity of ATP8A2 ( WT I376M ) ‐ CDC50A .

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B The effect of increasing levels of PS on the ATPase activity of ATP8A2 (WT and I376M)‐CDC50A complex.


C NBD ‐ labeled PS transport ( flippase ) activity of ATP8A2 ( WT I376M ) ‐ CDC50A . of NBD ‐ lipid flipped was shown .

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C NBD‐labeled PS transport (flippase) activity of ATP8A2 (WT and I376M)‐CDC50A complex. The percentage of NBD‐lipid flipped was shown.


D were treated with ATP8A1 siRNA1 for 72 h . ATP8A2 ‐ 1D4 ( WT or mutants ) myc ‐ CDC50A were transfected 48 h after siRNA transfection . were then stained for EHD1 1D4 ‐ tag . Scale bars , 10 μm .

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D Cells were treated with ATP8A1 siRNA1 for 72 h. ATP8A2‐1D4 (WT or mutants) and myc‐CDC50A were transfected 48 h after siRNA transfection. The cells were then stained for EHD1 and 1D4‐tag. Scale bars, 10 μm.


E of percentages of with perinuclear EHD1 localization . Data represent mean ± SD of three or four independent experiments . At least 20 were counted per experiment .

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E Quantification of percentages of cells with perinuclear EHD1 localization. Data represent mean ± SD of three or four independent experiments. At least 20 cells were counted per experiment.


F were treated with ATP8A1 siRNA for 72 h . GFP ‐ ATP8A2 ( WT or mutant ) myc ‐ CDC50A were transfected 48 h after siRNA transfection . were then stained for TfnR . Data represent mean ± SD of three or four independent experiments . At least 20 were counted per experiment .

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F Cells were treated with ATP8A1 siRNA for 72 h. GFP‐ATP8A2 (WT or mutant) and myc‐CDC50A were transfected 48 h after siRNA transfection. The cells were then stained for TfnR. Data represent mean ± SD of three or four independent experiments. At least 20 cells were counted per experiment.


G , H Surface biotinylation with Atp8a2 + / + or Atp8a2 − / − cortical neurons . of surface to total TfnR was quantified normalized to Atp8a2 + / + . Data represent mean value ( 2 mice for each genotype ) .

G, H Surface biotinylation assay with Atp8a2+/+ or Atp8a2−/− cortical neurons. The ratio of surface to total TfnR was quantified and normalized to Atp8a2+/+. Data represent mean value (2 mice for each genotype).


c d . Representation of in epidermal ( e ) cortical ( c ) files . Optical , longitudinal sections of 5 DAG old Col - 0 roots 12 HAT to ammonium ( C ) or nitrate ( D ) supplemented media . first 20 epidermal ( e ) cortex ( c ) ( from quiescent center ( QC ) ) are highlighted in grey in red on ammonium ( C ) , in blue green on nitrate ( D ) , respectively . Scale bar = 30 µm . Column bars denote geometric mean of lengths at respective positions . Lines represent a polynomial regression fit , with calculated slopes between 10 20 of 3 . 32639 + 0 . 17172 ( ammonium , epidermis ) , 1 . 22033 + 0 . 08754 ( ammonium , cortex ) 1 . 70502 + 0 . 09532 ( nitrate , epidermis ) , 0 . 82342 + 0 . 06973 ( nitrate , cortex ) . Data are derived from 3 biological replicates , n = 18 roots in each case .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and d. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG old Col-0 roots 12 HAT to ammonium (C) or nitrate (D) supplemented media. The first 20 epidermal (e) and cortex (c) cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (C), and in blue and green on nitrate (D), respectively. Scale bar=30 µm. Column bars denote the geometric mean of the cell lengths at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 3.32639+0.17172 (ammonium, epidermis), 1.22033+0.08754 (ammonium, cortex) and 1.70502+0.09532 (nitrate, epidermis), 0.82342+0.06973 (nitrate, cortex). Data are derived from 3 biological replicates, n=18 roots in each case.


a b . Maximum Z - stack projection of 5 DAG old roots expressing R2D2 auxin signaling reporter 12 HAT to ammonium ( a ) or nitrate ( b ) supplemented media . arrows mark position of 10th 20th from QC ; " e " " c " mark epidermis cortex , respectively . Scale bar = 50 µm . Graphs denote normalized relative auxin at respective positions . Lines represent polynomial regression fit with 95 % confidence band . Data are derived from 5 roots per condition from 3 biological replicates .

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

a and b. Maximum intensity Z-stack projection images of 5 DAG old roots expressing the R2D2 auxin signaling reporter 12 HAT to ammonium (a) or nitrate (b) supplemented media. White arrows mark the position of the 10th and 20th cells from QC; "e" and "c" mark epidermis and cortex, respectively. Scale bar=50 µm. Graphs denote normalized relative auxin levels at the respective positions. Lines represent polynomial regression fit with 95% confidence band. Data are derived from 5 roots per condition from 3 biological replicates.


b c . Representation of in epidermal ( e ) cortical ( c ) files . Optical , longitudinal sections of 5 DAG eir1 - 4 roots 12 HAT to ammonium ( B ) or nitrate ( C ) supplemented media . first 20 - 20 epidermal cortex ( from quiescent center ( QC ) ) are highlighted in grey in red on ammonium ( B ) in blue green on nitrate ( C ) , respectively . Scale bar = 30 µm . Column bars denote geometric mean of at respective positions . Lines represent a polynomial regression fit , with calculated slopes between 10 20 of 0 . 75884 + 0 . 02624 ( ammonium , epidermis ) , 1 . 13088 + 0 . 08446 ( ammonium , cortex ) 2 . 06912 + 0 . 10341 ( nitrate , epidermis ) , 0 . 99878 + 0 . 07278 ( nitrate , cortex ) . Data are derived from 3 biological replicates , n = 9 ( ammonium ) 8 ( nitrate ) roots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and c. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG eir1-4 roots 12 HAT to ammonium (B) or nitrate (C) supplemented media. The first 20-20 epidermal and cortex cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (B) and in blue and green on nitrate (C), respectively. Scale bar=30 µm. Column bars denote the geometric mean of cell length at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 0.75884+0.02624 (ammonium, epidermis), 1.13088+0.08446 (ammonium, cortex) and 2.06912+0.10341 (nitrate, epidermis), 0.99878+0.07278 (nitrate, cortex). Data are derived from 3 biological replicates, n=9 (ammonium) and 8 (nitrate) roots.


b . Higher magnification of pseudo - colored confocal of 5 DAG old roots expressing PIN2 - GFP 12 HAT to ammonium or nitrate supplemented media . " e " denotes epidermis " c " cortex , respectively . Color code represents GFP from low ( blue ) to high ( ) values . Scale bar = 12 µm . stars mark PIN2 - GFP localization on lateral membranes . Box plots display lateralization index ( signal detected on apical / basal membranes divided by signal value at inner / outer membranes ) of roots on ammonium ( n = 31 from 6 roots ) or nitrate ( n = 24 from 6 roots ) supplemented medium . statistical significance was evaluated with ANOVA at p < 0 . 05 . box chart components are defined as ; box ( 25 % - 75 % ) , central band ( median ) , central box ( mean ) range is within 1 . 5IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Higher magnification of pseudo-colored confocal images of 5 DAG old roots expressing PIN2-GFP 12 HAT to ammonium or nitrate supplemented media. "e" denotes epidermis and "c" cortex, respectively. Color code represents GFP intensity from low (blue) to high (white) values. Scale bar=12 µm. White stars mark PIN2-GFP protein localization on the lateral membranes. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes divided by the signal value at inner/outer membranes) of roots on ammonium (n=31 cells from 6 roots) or nitrate (n=24 cells from 6 roots) supplemented medium. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.


d . Microscopic showing PIN2 - Dendra signal five hours after photoconversion of PIN2 - Dendra into its red form . Depletion of red signal recovery of green signal over a 6 hours period was followed in parallel in 5 DAG old roots12 HAT to ammonium or nitrate supplemented media . Note increase in of green signal in roots transferred to nitrate . Graph represents mean signal + SD ( n = 6 roots per condition , 20 per root analyzed ) . experiment was repeated 3 times . Scale bar = 20 µm .

d. Microscopic images showing PIN2-Dendra fluorescent signal five hours after photoconversion of PIN2-Dendra into its red form. Depletion of the red signal and recovery of the green signal over a 6 hours period was followed in parallel in 5 DAG old roots12 HAT to ammonium or nitrate supplemented media. Note the increase in the intensity of the green signal in roots transferred to nitrate. Graph represents the mean signal + SD (n=6 roots per condition, 20 cells per root analyzed). The experiment was repeated 3 times. Scale bar=20 µm.


d . Box plots display distribution of derived PIN2 - GFP ( FI ) values ( in arbitrary units , a . u . ) in roots transferred to ammonium ( ( grey , epidermis ( ep ) red , cortex ( co ) to nitrate ( blue , epidermis ( ep ) green , cortex ( co ) ) . 5 per roots were analyzed in at least 9 roots per genotype per treatment . statistical significance was evaluated with ANOVA at p < 0 . 05 . box chart components are defined as ; box ( 25 % - 75 % ) , central band ( median ) , central box ( mean ) range is within 1 . 5IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Box plots display the distribution of the cell membrane derived PIN2-GFP fluorescence intensity (FI) values (in arbitrary units, a.u.) in roots transferred to ammonium ((grey, epidermis (ep) and red, cortex (co) and to nitrate (blue, epidermis (ep) and green, cortex (co)). 5 cells per roots were analyzed in at least 9 roots per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.


g . Box plots display lateralization index ( signal detected on apical / basal membranes vs inner / outer membranes ) of P2wt , P2D P2A roots transferred to ammonium ( grey ) or nitrate ( red ) supplemented medium . At least 24 from 5 roots were analyzed per genotype per treatment . statistical significance was evaluated with ANOVA at p < 0 . 05 . box chart components are defined as ; box ( 25 % - 75 % ) , central band ( median ) , central box ( mean ) range is within 1 . 5IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes vs inner/outer membranes) of P2wt, P2D and P2A roots transferred to ammonium (grey) or nitrate (red) supplemented medium. At least 24 cells from 5 roots were analyzed per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.


Immunofluorescence of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre ; ranklfl / fl mice ( Rankl - / - ) ranklfl / fl mice ( Rankl + / + ) . Scale bar = 50 μm .

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Immunofluorescence staining of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre; ranklfl/fl mice (Rankl-/-) and ranklfl/fl mice (Rankl+/+). Scale bar=50 μm.


Micro - CT statistical analyses of BMD , BV / TV , Tb . N Tb . Sp in trabecular bone from AdipoqCre ; ranklfl / fl ( Rankl - / - ) ranklfl / fl ( Rankl + / + ) mice at 4 , 8 16 ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t - ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Micro-CT and statistical analyses of BMD, BV/TV, Tb.N and Tb.Sp in trabecular bone from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 4 weeks, 8 weeks and 16 weeks (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t-test (** indicates P < 0.01), error bars are standard deviations.


Representative of TRAP of femur from AdipoqCre ; ranklfl / fl ( Rankl - / - ) ranklfl / fl ( Rankl + / + ) mice at 8 . Scale bar = 200 μm . Panel a1 a4 indicate trabecular bone , a2 a5 stand for growth plate , a3 a6 denote cortical bone .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of TRAP staining of femur from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 8 weeks. Scale bar=200 μm. Panel a1 and a4 indicate trabecular bone, a2 and a5 stand for growth plate, a3 and a6 denote cortical bone.


Immunofluorescence of osteocalcin from 8 - week - old mice ( n = 6 mice in Rankl - / - group 4 mice in Rankl + / + group ) . Scale bar = 50 μm . TB : trabecular bone . Statistical analyses of Ocn + ( n = 6 independent microscopic vision fields from random mice samples ) . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Immunofluorescence staining of osteocalcin from 8-week-old mice (n=6 mice in Rankl-/- group and 4 mice in Rankl+/+ group). Scale bar=50 μm. TB: trabecular bone. Statistical analyses of Ocn+ cells (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


Calcein of trabecular bone at 8 . Scale bar = 50 μm . TB : trabecular bone . are representative of 6 independent biological replicates in Rankl - / - 5 replicates in Rankl + / + group . Mineral apposition rate ( MAR ) of trabecular bone at 8 . ( n = 6 mice in Rankl - / - group 5 mice in Rankl + / + group ) . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Calcein staining of trabecular bone at 8 weeks. Scale bar=50 μm. TB: trabecular bone. Images are representative of 6 independent biological replicates in Rankl-/- and 5 replicates in Rankl+/+ group. Mineral apposition rate (MAR) of trabecular bone at 8 weeks.(n=6 mice in Rankl-/- group and 5 mice in Rankl+/+ group). Data were compared using an unpaired t‐test ( ** indicates P < 0.01), error bars are standard deviations.


Serum CTX - 1 , OCN bone marrow RANKL from Rankl - / - Rankl + / + mice at 4 ( n = 5 5 ) , 8 ( n = 5 6 ) 16 ( n = 5 5 ) . Data were compared using an unpaired t ‐ ( * indicates P < 0 . 05 , * * indicates P < 0 . 01 ) , error bars are standard deviations . .

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

Serum CTX-1, OCN and bone marrow RANKL levels from Rankl-/- and Rankl+/+ mice at 4 weeks (n=5 and 5), 8 weeks (n=5 and 6) and 16 weeks (n=5 and 5). Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations..


Micro - CT analyses of trabecular bone from Grounded Tail suspended model ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t ‐ ( * indicates P < 0 . 05 , * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Micro-CT analyses of trabecular bone from Grounded and Tail suspended model (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations.


Micro - CT analyses of sham OVX model of AdipoqCre ; ranklfl / fl ( Rankl - / - ) ranklfl / fl ( Rankl + / + ) mice ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Micro-CT analyses of sham and OVX model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


TRAP statistical analyses of numbers of osteoclasts in Sham OVX model ( are representative of 5 independent biological replicates in ( C ) , n = 5 independent microscopic vision fields in ( D ) ) . Scale bar = 100 µm . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

TRAP staining and statistical analyses of numbers of osteoclasts in Sham and OVX model (Images are representative of 5 independent biological replicates in (C), and n=5 independent microscopic vision fields in (D)). Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


Immunofluorescence of OCN statistical analyses of number of osteoblasts ( are representative of 5 independent biological replicates in ( E ) , n = 5 independent microscopic vision fields in ( F ) ) . Scale bar = 50 µm . TB : trabecular bone . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Immunofluorescence staining of OCN and statistical analyses of the number of osteoblasts (Images are representative of 5 independent biological replicates in (E), and n=5 independent microscopic vision fields in (F)). Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


Three - dimensional CT analyses of BMD , BV / TV , Tb . N , Tb . Sp in trabecular bone from Vehicle ( n = 5 independent biological replicates ) ROS ( n = 6 independent biological replicates ) model of AdipoqCre ; ranklfl / fl ( Rankl - / - ) ranklfl / fl ( Rankl + / + ) mice . Scale bar = 1 mm . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Three-dimensional CT analyses of BMD, BV/TV, Tb.N, and Tb.Sp in trabecular bone from Vehicle (n=5 independent biological replicates) and ROS (n=6 independent biological replicates) model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice. Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


TRAP statistical analyses of numbers of osteoclasts in trabecular bone from Vehicle ( n = 6 5 independent biological replicates ) ROS ( n = 6 6 independent biological replicates ) treatment . Scale bar = 100 µm . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations . n = 5 independent microscopic vision fields in ( D ) .

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

TRAP staining and statistical analyses of numbers of osteoclasts in the trabecular bone from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (D).


OCN statistical analyses of number of osteoblasts in femurs from Vehicle ( n = 6 5 independent biological replicates ) ROS ( n = 6 6 independent biological replicates ) treatment . Scale bar = 50 µm . TB : trabecular bone . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations . n = 5 independent microscopic vision fields in ( F ) .

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

OCN staining and statistical analyses of the number of osteoblasts in femurs from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (F).


( B ) Numbers of surface tumors per liver were counted . ( C ) cumulative diameter of surface tumors per liver per mouse reflects overall load .

O O O O 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) Numbers of surface tumors per liver were counted. (C) The cumulative diameter of all surface tumors per liver per mouse reflects the overall tumor load.


( D ) of livers including mass relative to ( LW / BW ) was recorded .

(D) Weight of the livers including tumor mass relative to the body weight (LW/BW ratio) was recorded.


( E ) Representative pictures of total livers with tumors of indicated genotypes .

(E) Representative pictures of total livers with tumors of the indicated genotypes.


( A ) Immunoblots of murine wt non - tumorous tissue lysates were probed for CASP2 , RAIDD , p53 . HSP90 PonceauS served as loading controls .

(A) Immunoblots of murine wt non-tumorous and tumor tissue lysates were probed for CASP2, RAIDD, and p53. HSP90 and PonceauS protein staining served as loading controls.


( B ) Proliferation of tissue was determined by of Ki67 - positive nuclei analyzed by flow cytometry . T : wt n = 17 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 8 ; NT : wt n = 12 , Casp - 2 - / - n = 11 , Raidd - / - n = 7 , Pidd1 - / - n = 5 . N - numbers refer to biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proliferation of tumor tissue was determined by the percentage of Ki67-positive nuclei analyzed by flow cytometry. T: wt n=17, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=8; NT: wt n=12, Casp-2-/- n=11, Raidd-/- n=7, Pidd1-/- n=5. N-numbers refer to biological replicates.


( D ) Immunoblots of experiment described in ( C ) at t ( 8h ) , t ( 48h ) , t ( weaning + 5d ) for CASP2 , RAIDD , p53 , γH2AX , HSP90 as loading control .

O O O O O O O O O O O O O O O O O O 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) Immunoblots of the experiment described in (C) at t(8h), t(48h), and t(weaning+5d) for CASP2, RAIDD, p53, γH2AX, and HSP90 as loading control.


( E ) number of binucleated hepatocytes per field was counted on H & E stained paraffin sections of PBS treated wt Pidd1 - / - mice at t ( 0h ) at t ( 48h ) after PBS or DEN treatment . N - numbers refer to biological replicates .

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

(E) The number of binucleated hepatocytes per field was counted on H&E stained paraffin sections of PBS treated wt and Pidd1-/- mice at t(0h) and at t(48h) after PBS or DEN treatment. N-numbers refer to biological replicates.


( F ) Immunofluorescence of same livers was perfomed for distal centriolar CP110 to stain centrioles , β - catenin for borders , DAPI to stain . Representative of a mononucleated hepatocyte with 2 centrioles a binucleated hepatocyte with 4 centrioles is 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

(F) Immunofluorescence of the same livers was perfomed for the distal centriolar protein CP110 to stain centrioles, β-catenin for cell borders, and DAPI to stain DNA. Representative images of a mononucleated hepatocyte with 2 centrioles and a binucleated hepatocyte with 4 centrioles is shown.


( G ) Centriole number was quantified with respect to number of nuclei per in wt Pidd1 - / - livers at time points described in ( E ) ; n = 3 biological replicates .

O O O O O O O O O O O O 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) Centriole number was quantified with respect to the number of nuclei per cell in wt and Pidd1-/- livers at the time points described in (E); n=3 biological replicates.


( A ) Binucleation was assessed blinded in H & E stained sections of murine non - ( NT ) tissue ( T ) T : wt n = 14 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 10 ; NT : wt n = 8 , Casp - 2 - / - n = 10 , Raidd - / - n = 8 , Pidd1 - / - n = 9 . N - numbers refer to biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Binucleation was assessed blinded in H&E stained sections of murine non-tumor (NT) and tumor tissue (T) T: wt n=14, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=10; NT: wt n=8, Casp-2-/- n=10, Raidd-/- n=8, Pidd1-/- n=9. N-numbers refer to biological replicates.


Binucleation was assessed blinded in H & E stained sections of murine non - ( NT ) tissue ( T ) also shown in ( B ) . T : wt n = 14 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 10 ; NT : wt n = 8 , Casp - 2 - / - n = 10 , Raidd - / - n = 8 , Pidd1 - / - n = 9 . N - numbers refer to biological replicates .

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Binucleation was assessed blinded in H&E stained sections of murine non-tumor (NT) and tumor tissue (T) also shown in (B). T: wt n=14, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=10; NT: wt n=8, Casp-2-/- n=10, Raidd-/- n=8, Pidd1-/- n=9. N-numbers refer to biological replicates.


C , D ( C ) Representative histograms of nuclei stained for , which were isolated from non - tissue of wt Casp2 - / - mice . These are marked as red " x " in shown in ( D ) were degree of polyploidy is reflected as of octaploid nuclei ( T : wt n = 17 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 8 ; NT : wt n = 12 , Casp - 2 - / - n = 11 , Raidd - / - n = 7 , Pidd1 - / - n = 5 ) . N - numbers refer to biological replicates .

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C, D (C) Representative histograms of nuclei stained for DNA, which were isolated from non-tumor and tumor tissue of wt and Casp2-/- mice. These animals are marked as red "x" in the quantification shown in (D) were the degree of polyploidy is reflected as percentage of octaploid nuclei (T: wt n=17, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=8; NT: wt n=12, Casp-2-/- n=11, Raidd-/- n=7, Pidd1-/- n=5). N-numbers refer to biological replicates.


Copy number variations were determined using whole genome sequencing of 30 nuclei " mini - bulks " isolated from Casp2 - / - or wt tumors ( n = 11 per genotype ) . Diploid tetraploid pools of nuclei were analyzed separately . A , B ( A ) Representative dot plots show degree of aneuploidy heterogeneity are quantified in ( B , empty symbols ) . Data are represented as median , statistical significance determined by one - way ANOVA with multiplicity correction ( Sidak - Holm ) ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 . N - numbers refer to biological replicates .

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

Copy number variations were determined using whole genome sequencing of 30 nuclei "mini-bulks" isolated from Casp2-/- or wt tumors (n=11 per genotype). Diploid and tetraploid pools of tumor cell nuclei were analyzed separately. A, B (A) Representative dot plots show the degree of aneuploidy and heterogeneity and are quantified in (B, empty symbols). Data are represented as median, statistical significance determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01. N-numbers refer to biological replicates.


( A ) Matched human non - ( NT ) tissue ( T ) samples were analyzed by immunoblotting for CASP2 , PIDD1 RAIDD on membranes a b . PonceauS serves as loading control . asterisk refers to an unspecific band , " C " " CC " indicate functionally active fragments of PIDD1 generated by autoprocessing , PIDD - C PIDD - CC . Densitometric of these blots is shown in Fig . EV4A .

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(A) Matched human patient non-tumor (NT) and tumor tissue (T) samples were analyzed by immunoblotting for CASP2, PIDD1 and RAIDD on membranes a and b. PonceauS protein staining serves as loading control. The asterisk refers to an unspecific band, "C" and "CC" indicate the functionally active fragments of PIDD1 generated by autoprocessing, PIDD-C and PIDD-CC. Densitometric quantification of these blots is shown in Fig. EV4A.


( B ) mRNA expression of PIDDosome components in TCGA Provisional LIHC data set across disease stages . are divided on p53 mutation / deletion ( mut / del ) status . Statistical significance was determined using two - way ANOVA with Tukey ' s post - hoc . central band represents median , boxes indicate 25th 75th percentile , whiskers are 5th 95th percentiles , statistical significance was defined as * p ≤ 0 . 05 , * * p ≤ 0 . 01 .

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(B) mRNA expression analysis of the PIDDosome components in the TCGA Provisional LIHC data set across disease stages. Patients are divided based on the p53 mutation/deletion (mut/del) status. Statistical significance was determined using two-way ANOVA with Tukey's post-hoc analysis. The central band represents the median, the boxes indicate the 25th and 75th percentile, the whiskers are the 5th and 95th percentiles, statistical significance was defined as * p≤0.05, ** p≤0.01.


( C ) same data set was analyzed for recurrence - free survival on high or low expression of CASP2 , RAIDD or PIDD1 divided at median . Statistical significance was determined using a Log - rank .

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(C) The same data set was analyzed for recurrence-free survival based on high or low expression of CASP2, RAIDD or PIDD1 divided at the median. Statistical significance was determined using a Log-rank test.


( B ) E2F target genes expression was analyzed for correlation with expression of CASP2 , RAIDD PIDD1 using Spearman ' s correlation coefficient . central band represents median , boxes indicate 2nd 3rd quartile , whiskers are 1 . 5 times interquartile range ( n = 200 genes ) .

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(B) E2F target genes expression was analyzed for correlation with expression of CASP2, RAIDD and PIDD1 using Spearman's correlation coefficient. The central band represents the median, the boxes indicate the 2nd and 3rd quartile, and the whiskers are 1.5 times the interquartile range (n=200 genes).


Correlation ( Pearson ) of CASP2 mRNA with transcript of Ki67 in TCGA data set .

Correlation (Pearson) of CASP2 mRNA with transcript levels of Ki67 in the TCGA data set.


Correlation ( Pearson ) of RAIDD PIDD1 mRNA with transcript of Ki67 in TCGA data set .

Correlation (Pearson) of RAIDD and PIDD1 mRNA with transcript levels of Ki67 in the TCGA data set.


( A ) ploidy index is inferred as 1 / density of tumorous ( T ) non - tumorous ( NT ) tissue of HCC with different liver diseases . ALD - alcoholic liver disease ( n = 65 ) ; HBV - hepatitis B infection ( n = 24 ) ; HCV - hepatitis C infection ( n = 85 ) ; NASH - non - alcoholic steatohepatitis ( n = 33 ) . Statistical significance was determined by one - way ANOVA with multiplicity correction ( Sidak - Holm ) ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . n - numbers refer to number of samples analyzed .

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(A) The ploidy index is inferred as 1/cell density of tumorous (T) and non-tumorous (NT) tissue of HCC patients with different liver diseases. ALD - alcoholic liver disease (n=65); HBV - hepatitis B virus infection (n=24); HCV - hepatitis C virus infection (n=85); NASH - non-alcoholic steatohepatitis (n=33). Statistical significance was determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01, *** p≤0.001. n-numbers refer to the number of patient samples analyzed.


( B ) were grouped according to HCC density as surrogate for ploidy . Data were divided at median exact numbers are shown in graphs . Median recurrence - free survival ( RFS ) for a period of 10 years for with low ploidy was 4 . 8 years . For with high ploidy median RFS could not be defined as more than 50 % of survived without relapse . characteristics are shown in Appendix table S1 . A Log - rank was used to determine statistical significance .

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(B) All patients were grouped according to HCC tumor cell density as surrogate for tumor ploidy. Data were divided at the median and exact patient numbers are shown in the graphs. Median recurrence-free survival (RFS) for a period of 10 years for patients with low tumor ploidy was 4.8 years. For patients with high tumor ploidy the median RFS could not be defined as more than 50% of the patients survived without relapse. Patient characteristics are shown in Appendix table S1. A Log-rank test was used to determine statistical significance.


A Baseline blood of granulocytes . One - way ANOVA ; n = 8 mice per ZT ; * P = 0 . 0001 ZT5 vs . ZT13 .

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A Baseline blood counts of granulocytes. One-way ANOVA; n = 8 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.


B Flow cytometry of neutrophils in digested hearts . representative dot plots show gating strategy for cardiacneutrophils ( CD45 + Ly6G + CD11b + ) at ZT5 . One - way ANOVA ; n = 5 mice per ZT ; * P = 0 . 0001 ZT5 vs . ZT13 .

B Flow cytometry quantification of neutrophils in digested hearts. The representative dot plots show the gating strategy for cardiacneutrophils (CD45+Ly6G+CD11b+) at ZT5. One-way ANOVA; n = 5 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.


C Representative immunostainings for neutrophils in myocardium ( left ventricle ) , identified as Ly6G - positive ( 20x magnification ) .

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C Representative immunostainings for neutrophils in the myocardium (left ventricle), identified as Ly6G-positive (20x magnification).


D Cardiac mRNA expression normalized to HPRT . One - way ANOVA ; n = 5 mice per ZT ; ZT5 vs . ZT17 : * P = 0 . 0064 ( CXCL1 ) , * P = 0 . 0007 ( CXCL2 ) , * P = 0 . 0007 ( CXCL5 ) , * P = 0 . 0001 ( ICAM - 1 ) , * P = 0 . 0009 ( VCAM - 1 ) , * P = 0 . 0181 ( CCL3 ) , * P = 0 . 0360 ( CCL5 ) .

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D Cardiac mRNA expression levels normalized to HPRT. One-way ANOVA; n = 5 mice per ZT; ZT5 vs. ZT17: *P = 0.0064 (CXCL1), *P = 0.0007 (CXCL2), *P = 0.0007 (CXCL5), *P = 0.0001 (ICAM-1), *P = 0.0009 (VCAM-1), *P = 0.0181 (CCL3), *P = 0.0360 (CCL5).


E Mean ( MFI ) of CXCR2 expression by neutrophils in blood heart . One - way ANOVA ; n = 3 mice for ZT1 , ZT17 , ZT21 n = 5 for ZT5 , ZT9 , ZT13 ; ZT5 vs . ZT13 : * P = 0 . 0425 ( blood ) , * P = 0 . 0078 ( heart ) .

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E Mean fluorescence intensity (MFI) of CXCR2 expression by neutrophils in blood and heart. One-way ANOVA; n = 3 mice for ZT1, ZT17, ZT21 and n = 5 for ZT5, ZT9, ZT13; ZT5 vs. ZT13: *P = 0.0425 (blood), *P = 0.0078 (heart).


A Flow cytometry of neutrophils in hearts representative immunostainings for neutrophils in infarct , identified as Ly6G - positive ( 5x 20x magnifications ) . Two - way ANOVA followed by Bonferroni post - hoc ; n = 5 mice for no MI , n = 5 for 12h post MI , n = 3 for 24h post MI n = 3 for 72h post MI in both ZT groups ; ZT5 vs . ZT13 : * P = 0 . 0161 ( 12h MI ) , * P = 0 . 003 ( 24h MI ) .

A Flow cytometry analysis of neutrophils in hearts and representative immunostainings for neutrophils in the infarct area, identified as Ly6G-positive (5x and 20x magnifications). Two-way ANOVA followed by Bonferroni post-hoc test; n=5 mice for no MI, n = 5 for 12h post MI, n = 3 for 24h post MI and n = 3 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0161 (12h MI), *P = 0.003 (24h MI).


B Flow cytometry of neutrophils in bone marrow . Two - way ANOVA followed by Bonferroni post - hoc ; n = 5 mice for no MI , n = 5 for 12h post MI , n = 4 for 24h post MI n = 4 for 72h post MI in both ZT groups ; ZT5 vs . ZT13 : * P = 0 . 0471 ( 12h MI ) , * P = 0 . 0035 ( 24h MI ) .

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B Flow cytometry analysis of neutrophils in bone marrow. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice for no MI, n = 5 for 12h post MI, n = 4 for 24h post MI and n = 4 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0471 (12h MI), *P = 0.0035 (24h MI).


C Blood of granulocytes . Two - way ANOVA followed by Bonferroni post - hoc ; n = 9 mice for no MI , n = 10 for 12h post MI , n = 13 for 24h post MI n = 10 for 72h post MI in both ZT groups ; ZT5 vs . ZT13 : * P = 0 . 0364 ( 24h MI ) .

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C Blood counts of granulocytes. Two-way ANOVA followed by Bonferroni post-hoc test; n= 9 mice for no MI, n = 10 for 12h post MI, n = 13 for 24h post MI and n = 10 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0364 (24h MI).


D Flow cytometry of neutrophils in hearts bone marrow under inverted cycle conditions . Two - way ANOVA followed by Bonferroni post - hoc ; n = 3 mice for no MI in both ZT groups , n = 5 for ZT5 n = 6 for ZT13 24h post MI ; ZT5 vs . ZT13 : * P = 0 . 0147 ( 24h MI ) .

D Flow cytometry analysis of neutrophils in hearts and bone marrow under inverted light cycle conditions. Two-way ANOVA followed by Bonferroni post-hoc test; n = 3 mice for no MI in both ZT groups, n = 5 for ZT5 and n = 6 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0147 (24h MI).


E Representative gating strategy for GMPs in bone marrow , identified as identified as lineage negative ( CD11b - , Gr1 - , B220 - , CD3 - Ter119 - ) Sca - 1 - , c - kit + , CD16 / 32 + , CD34 + . Flow cytometry of GMP in bone marrow . Two - way ANOVA followed by Bonferroni post - hoc ; n = 4 mice for no MI 24h post MI in both groups ; ZT5 vs . ZT13 : * P = 0 . 0077 ( no MI ) , * P = 0 . 0013 ( 24h MI ) .

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E Representative gating strategy for GMPs in the bone marrow, identified as identified as lineage negative (CD11b-, Gr1-, B220-, CD3- and Ter119-) and Sca-1-, c-kit+, CD16/32+, CD34+. Flow cytometry quantification of GMP in the bone marrow. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for no MI and 24h post MI in both groups; ZT5 vs. ZT13: *P = 0.0077 (no MI), *P = 0.0013 (24h MI).


F CXCL12 in bone marrow lavage . Two - way ANOVA followed by Bonferroni post - hoc ; n = 7 mice for no MI n = 6 for 24h post MI in both groups ; ZT5 vs . ZT13 : * P = 0 . 0077 ( no MI ) , * P = 0 . 0013 ( 24h MI ) .

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F CXCL12 levels in bone marrow lavage. Two-way ANOVA followed by Bonferroni post-hoc test; n = 7 mice for no MI and n = 6 for 24h post MI in both groups; ZT5 vs. ZT13: *P = 0.0077 (no MI), *P = 0.0013 (24h MI).


G Plasma of pro - inflammatory cytokines chemokines . Two - way ANOVA followed by Bonferroni post - hoc ; n = 11 mice for no MI in both ZT groups , n = 7 for ZT5 n = 8 for ZT13 24h post MI ; ZT5 vs . ZT13 : * P = 0 . 0271 ( CXCL12 , no MI ) , * P = 0 . 0108 ( TNFα , 24h MI ) , * P = 0 . 001 ( G - CSF , 24h MI ) , * P = 0 . 005 ( CXCL1 , 24h MI ) , * P = 0 . 0005 ( CXCL2 , 24h MI ) , * P = 0 . 0016 ( CCL3 24h MI ) , * P = 0 . 0144 ( CCL5 , 24h MI ) .

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G Plasma levels of pro-inflammatory cytokines and chemokines. Two-way ANOVA followed by Bonferroni post-hoc test; n = 11 mice for no MI in both ZT groups, n = 7 for ZT5 and n = 8 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0271 (CXCL12, no MI), *P = 0.0108 (TNFα, 24h MI), *P = 0.001 (G-CSF, 24h MI), *P = 0.005 (CXCL1, 24h MI), *P = 0.0005 (CXCL2, 24h MI), *P = 0.0016 (CCL3 24h MI), *P = 0.0144 (CCL5, 24h MI).


A Permanent LAD occlusion was perfomed at ZT5 or ZT13 . TTC ( , infarct ; red , vital myocardium ) of infarct normalized to left ventricle ( LV ) . Student ` s t - ; n = 4 mice for ZT5 n = 5 for ZT13 MI ; * P = 0 . 0041 .

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A Permanent LAD occlusion was perfomed at ZT5 or ZT13. TTC staining (white, infarct; red, vital myocardium) and quantification of infarct size normalized to the left ventricle (LV). Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0041.


B Plasma troponin I 24 hours after MI . Student ` s t - ; n = 4 mice for ZT5 n = 5 for ZT13 MI ; * P = 0 . 0007 .

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B Plasma troponin I levels 24 hours after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0007.


C Flow cytometry of dead cardiomyocytes ( CD45 - , zombie + ) 24 hours after MI . Student ` s t - ; n = 3 mice in both groups ; * P = 0 . 0072 .

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C Flow cytometry analysis of dead cardiomyocytes (CD45-, zombie+) 24 hours after MI. Student`s t-test; n = 3 mice in both groups; *P = 0.0072.


D Survival rates after MI cause of . Log - rank ; n = 87 mice in both groups ; * P = 0 . 0006 .

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D Survival rates after MI and cause of death. Log-rank test; n = 87 mice in both groups; *P = 0.0006.


E Myofibroblasts within infarcts were quantified by alpha smooth muscle actin ( αSMA ) as between stained total of random fields . Student ` s t - ; n = 4 mice in both groups ; * P = 0 . 0134 .

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E Myofibroblasts within infarcts were quantified by alpha smooth muscle actin (αSMA) staining as ratio between stained and total area of random fields. Student`s t-test; n = 4 mice in both groups; *P = 0.0134.


F Masson ´ s trichome of fibrosis ( blue , collagen ; red , vital myocardium ) relative to total LV ( * P = 0 . 0095 ) as well as LV anterior wall thickness ( * P = 0 . 0068 ) 7 days after MI . Student ` s t - ; n = 6 mice for ZT5 n = 7 for ZT13 MI .

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F Masson´s trichome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total LV (*P = 0.0095) as well as LV anterior wall thickness (*P = 0.0068) 7 days after MI. Student`s t-test; n = 6 mice for ZT5 and n = 7 for ZT13 MI.


G of relative collagen type I content identified by Sirius Redstaining 7 days after MI . Student ` s t - ; n = 4 mice for ZT5 n = 5 for ZT13 MI ; * P = 0 . 0175 .

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G Analysis of relative collagen type I content identified by Sirius Redstaining 7 days after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0175.


H Echocardiography assessment of ejection fraction ( EF ) , end - systolic - volume ( ESV ) end - diastolic - volume ( EDV ) . Two - way ANOVA ; n = 6 mice for no MI for both groups , n = 6 for ZT5 n = 9 for ZT13 at 72h post MI , n = 8 for both groups at 7d post MI n = 7 for ZT5 n = 5 for ZT13 14d post MI ; ZT5 vs . ZT13 : * P = 0 . 0001 ( EF , 3d ) , * P = 0 . 0042 ( EF , 7d ) , * P = 0 . 0121 ( EF , 14d ) ; * P = 0 . 0253 ( ESV , 3d ) , * P = 0 . 0421 ( ESV , 7d ) , * P = 0 . 0005 ( ESV , 14d ) ; * P = 0 . 0053 ( EDV , 14d ) .

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H Echocardiography assessment of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV). Two-way ANOVA; n = 6 mice for no MI for both groups, n = 6 for ZT5 and n = 9 for ZT13 at 72h post MI, n = 8 for both groups at 7d post MI and n = 7 for ZT5 and n = 5 for ZT13 14d post MI; ZT5 vs. ZT13: *P = 0.0001 (EF, 3d), *P = 0.0042 (EF, 7d), *P = 0.0121 (EF, 14d); *P = 0.0253 (ESV, 3d), *P = 0.0421 (ESV, 7d), *P = 0.0005 (ESV, 14d); *P = 0.0053 (EDV, 14d).


B Flow cytometry of cardiacneutrophils 24 hours after ZT13 MI . dotted indicates cardiacneutrophil 24h after ZT5 MI , shown in Fig . 2A . Student ` s t - ; n = 3 mice for isotype n = 5 for Ly6G injected mice ; * P = 0 . 0004 .

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B Flow cytometry analysis of cardiacneutrophils 24 hours after ZT13 MI. The dotted line indicates cardiacneutrophil counts 24h after ZT5 MI, shown in Fig. 2A. Student`s t-test; n = 3 mice for isotype and n = 5 for Ly6G injected mice; *P = 0.0004.


C Plasma TNFα 24 hours after ZT13 MI . Student ` s t - . n = 8 independent samples for isotype n = 6 for Ly6G injected mice ; * P = 0 . 0019 .

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C Plasma TNFα levels 24 hours after ZT13 MI. Student`s t-test. n=8 independent samples for isotype and n=6 for Ly6G injected mice; *P = 0.0019.


D Infarct relative to left ventricular ( LV ) plasmatroponin I 24 hours after ZT5 or ZT13 MI . Student ` s t - ; For infarct n = 3 mice in both groups for ZT5 n = 4 mice for isotype n = 3 mice for Ly6G at ZT13 ; Isotype vs . Ly6G : * P = 0 . 0158 ( ZT13 ) . For troponin n = 8 mice for isotype n = 6 mice for Ly6G with ZT13 MI ; * P = 0 . 0012 .

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D Infarct size relative to left ventricular area (LV) and plasmatroponin I levels 24 hours after ZT5 or ZT13 MI. Student`s t-test; For infarct size n = 3 mice in both groups for ZT5 and n = 4 mice for isotype and n = 3 mice for Ly6G at ZT13; Isotype vs. Ly6G: *P = 0.0158 (ZT13). For troponin levels n = 8 mice for isotype and n = 6 mice for Ly6G with ZT13 MI; *P = 0.0012.


E Masson ' s - trichrome of fibrosis ( blue , collagen ; red , vital myocardium ) relative to total of LV ( * P = 0 . 0084 ) ; morphometric of LV anterior wall thickness ( * P = 0 . 0027 ) 7 days after ZT13 MI . Student ` s t - ; n = 5 mice for isotype n = 4 mice for Ly6G .

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E Masson's-trichrome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total area of the LV (*P = 0.0084); morphometric quantification of the LV anterior wall thickness (*P = 0.0027) 7 days after ZT13 MI. Student`s t-test; n = 5 mice for isotype and n = 4 mice for Ly6G.


F of collagen type I within infarcts identified by Sirius Red 7 days after ZT13 MI . Student ` s t - ; n = 5 mice for isotype n = 4 mice for Ly6G ; * P = 0 . 0375 .

F Analysis of collagen type I fibers within infarcts identified by Sirius Red staining 7 days after ZT13 MI. Student`s t-test; n = 5 mice for isotype and n = 4 mice for Ly6G; *P = 0.0375.


G Echocardiography measurement of ejection fraction ( EF ) , end - systolic - volume ( ESV ) end - diastolic - volume ( EDV ) before after ZT13 MI . Two - way ANOVA ; n = 6 mice for no MI in both groups , n = 9 for isotype n = 5 for Ly6G at 72h post MI , n = 8 for isotype n = 4 for Ly6G at 7d post MI . Isotype vs . Ly6G : * P = 0 . 0001 ( EF , 3d ) , * P = 0 . 0227 ( EF , 7d ) ; MI * P = 0 . 0133 ( ESV , 3d ) , * P = 0 . 0004 ( ESV , 7d ) ; * P = 0 . 0017 ( EDV , 7d ) .

G Echocardiography measurement of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV) before and after ZT13 MI. Two-way ANOVA; n = 6 mice for no MI in both groups, n = 9 for isotype and n = 5 for Ly6G at 72h post MI, n = 8 for isotype and n = 4 for Ly6G at 7d post MI. Isotype vs. Ly6G: *P = 0.0001 (EF, 3d), *P = 0.0227 (EF, 7d); MI *P = 0.0133 (ESV, 3d), *P = 0.0004 (ESV, 7d); *P = 0.0017 (EDV, 7d).


A Representative flow cytometry of CXCR2highneutrophils in blood after ZT5 ZT13 MI . Two - way ANOVA followed by Bonferroni post - hoc ; n = 4 mice for time points in both groups ; ZT5 vs . ZT13 : * P = 0 . 0001 ( 4h ) , * P = 0 . 0001 ( 8h ) , * P = 0 . 0447 ( 12h ) .

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A Representative flow cytometry analysis and quantification of CXCR2highneutrophils in the blood after ZT5 and ZT13 MI. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for all time points in both groups; ZT5 vs. ZT13: *P = 0.0001 (4h), *P = 0.0001 (8h), *P = 0.0447 (12h).


B mean ( MFI ) of CXCX2 expression by cardiacneutrophils 12h post - MI after ZT5 ZT13 MI . Student ` s t - ; n = 4 mice in both groups ; * P = 0 . 0001 .

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B Percentage and mean fluorescence intensity (MFI) of CXCX2 expression by cardiacneutrophils 12h post-MI after ZT5 and ZT13 MI. Student`s t-test; n = 4 mice in both groups; *P = 0.0001.


C of CXCR2highneutrophils in blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc ; n = 4 mice in both groups ; DMSO vs . SB225002 : * P = 0 . 0005 ( ZT13 ) .

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C Percentage of CXCR2highneutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0005 (ZT13).


D Flow cytometry of neutrophils in hearts 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc ; n = 4 mice in both groups ; DMSO vs . SB225002 : * P = 0 . 0079 ( ZT13 ) .

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D Flow cytometry quantification of neutrophils in hearts 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0079 (ZT13).