( B ) Fluorescent miR - 21 - 3p in situ hybridization ( pink ) in dorsal skin of acutely irradiated ( Ac - UV ) and non - irradiated ( no UV ) Ppard + / + and - / - mice . E : epidermis ; D : dermis ; HF : hair follicle . Magnification bar :

O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O B-TISSUE O O O B-TISSUE O O O B-TISSUE I-TISSUE O O O O

(B) Fluorescent miR-21-3p in situ hybridization (pink) in dorsal skin of acutely irradiated (Ac-UV) and non-irradiated (no UV) Ppard +/+ and -/- mice. E: epidermis; D: dermis; HF: hair follicle. Magnification bar: 100m


( C ) RT - qPCR quantification of relative pri - miR - 21 and miR - 21 - 3p levels in the epidermis ( left and middle ) , and of relative miR - 21 - 3p in the dermis ( right ) of acutely irradiated ( Ac - UV ; + ) and non - irradiated ( - ) Ppard + / + and - / - mice . Pri - miR - 21 : Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 022 ; miR - 21 - 3p : Ppard + / + no UV vs Ppard + / + Ac - UV P = 0 . 017 , Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 008 . N = 3 - 4 animals per group , one representative experiment is shown out of three independent replicates .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) RT-qPCR quantification of relative pri-miR-21 and miR-21-3p levels in the epidermis (left and middle), and of relative miR-21-3p in the dermis (right) of acutely irradiated (Ac-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. Pri-miR-21: Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.022; miR-21-3p: Ppard +/+ no UV vs Ppard +/+ Ac-UV P = 0.017, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.008. N=3-4 animals per group, one representative experiment is shown out of three independent replicates.


( D ) RT - qPCR quantification of relative miR - 21 - 3p levels in total skin of chronically irradiated ( Chr - UV ; + ) and non - irradiated ( - ) Ppard + / + and - / - mice . miR - 21 - 3p : Ppard + / + no UV vs Ppard + / + Chr - UV P = 0 . 008 , Ppard + / + Chr - UV vs Ppard - / - Chr - UV P = 0 . 002 , ns : non significant . N = 4 animals per groups , one representative experiment is shown out of two independent replicates .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) RT-qPCR quantification of relative miR-21-3p levels in total skin of chronically irradiated (Chr-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. miR-21-3p: Ppard +/+ no UV vs Ppard +/+ Chr-UV P = 0.008, Ppard +/+ Chr-UV vs Ppard -/- Chr-UV P = 0.002, ns: non significant. N=4 animals per groups, one representative experiment is shown out of two independent replicates.


( 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

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

(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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE 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 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 .

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

(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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 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 .

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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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-21-5p 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 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 .

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

O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 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 .

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

(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 + / + and - / - mice . Loading control : GAPDH .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O B-GENEPROD O

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


( 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 .

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

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

(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 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 .

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

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

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

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

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

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

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O

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

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

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 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 .

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

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 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 .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells co ‐ transfected with GFP ‐ ATP8A1 and 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 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 .

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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 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 .

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 and E191Q ) ‐ CDC50A complex . The percentage 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 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 .

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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 ‐ 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 .

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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 ‐ 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 .

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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 , 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 .

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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 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 .

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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 cells were transiently transfected with GFP ‐ lact ‐ C2 and 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 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 .

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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 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 .

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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 cells co ‐ transfected with GFP ‐ ATP8A2 ( WT ) and myc ‐ CDC50A were stained for myc ‐ tag and 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 The effect of increasing levels of PS on the ATPase activity of ATP8A2 ( WT and I376M ) ‐ CDC50A complex .

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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 and I376M ) ‐ CDC50A complex . The percentage 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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O B-TISSUE O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O 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-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-TISSUE 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 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 .

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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 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 .

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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 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 .

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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 ( 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 .

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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 staining of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre ; ranklfl / fl mice ( Rankl - / - ) and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 . .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 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 ) .

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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 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 ) .

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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 ) The cumulative diameter of all surface tumors per liver per mouse reflects the overall tumor load .

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

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(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 the indicated genotypes .

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


( 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 .

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(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 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 .

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

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(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 ) 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 .

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

O O O B-EXP_ASSAY O O O B-TISSUE O O O O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-SUBCELLULAR 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 the number of nuclei per cell in wt and Pidd1 - / - livers at the time points described in ( E ) ; n = 3 biological replicates .

O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD O O O B-TISSUE 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 - 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 .

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

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

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 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 .

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

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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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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 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 .

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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 ) 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 .

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

(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 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 ) .

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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 levels of Ki67 in the TCGA data set .

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


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

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