( A , B ) Representative immunofluorescence microscopy of distribution of septin 7 ( red ) , Cdc42 ( red ) tubulin ( green ) in LT - HSCs from borg4fl / fl or borg4 ∆ / ∆ mice . Nuclei were stained with DAPI ( blue ) , scale bar = 5 µm . ( C ) of with a polar distribution of septin 7 , Cdc42 or tubulin in borg4fl / fl or borg4 ∆ / ∆ LT - HSCs . At least 3 biological repeats , at least 50 were scored per sample . Error bars indicate mean ± , Two - way ANOVA , * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 .

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

(A,B) Representative immunofluorescence microscopy images of the distribution of septin 7 (red), Cdc42 (red) and tubulin (green) in LT-HSCs from borg4fl/fl or borg4∆/∆ mice. Nuclei were stained with DAPI (blue), scale bar= 5 µm. (C) Percentage of cells with a polar distribution of septin 7, Cdc42 or tubulin in borg4fl/fl or borg4∆/∆ LT-HSCs. At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, ***P<0.001, **P<0.01, *P<0.05.


( D , E ) Representative immunofluorescence microscopy of distribution of borg4 ( red ) , Cdc42 ( red ) or tubulin ( green ) in LT - HSCs from septin 7fl / fl or septin 7 ∆ / ∆ mice . Nuclei were stained with DAPI ( blue ) . scale bar = 5 µm . ( F ) of with a polar distribution of borg4 , Cdc42 or tubulin in septin 7fl / fl or septin 7 ∆ / ∆ LT - HSCs . At least 3 biological repeats , at least 30 to 50 were scored per sample . Error bars indicate mean ± , Two - way ANOVA , * * P < 0 . 01 , * P < 0 . 05 .

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

(D,E) Representative immunofluorescence microscopy images of the distribution of borg4 (red), Cdc42 (red) or tubulin (green) in LT-HSCs from septin 7fl/fl or septin 7∆/∆ mice. Nuclei were stained with DAPI (blue). scale bar= 5 µm. (F) Percentage of cells with a polar distribution of borg4, Cdc42 or tubulin in septin 7fl/fl or septin 7∆/∆ LT-HSCs. At least 3 biological repeats, at least 30 to 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, **P<0.01, *P<0.05.


( J - O ) of donor derived ( borg4fl / fl or borg4 ∆ / ∆ or septin 7fl / fl or septin 7 ∆ / ∆ ) B - ( B220 positive ) , T - ( CD3 positive ) myeloid ( Mac - 1 + or Gr + positive ) among BM , of donor derived LT - HSCs , ST - HSCs LMPPs in LSK of donor derived CLPs in lin - Sca1C - kit low in at post - transplantation ( n = 12 ) . Error bars indicate mean ± SD , * * * * P < 0 . 0001 , * * P < 0 . 01 * P < 0 . 05 , Two - way ANOVA 2 unpaired t - .

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

(J-O) Percentage of donor derived (borg4fl/fl or borg4∆/∆ or septin 7fl/fl or septin 7∆/∆) B-cells (B220 positive cells), T-cells (CD3 positive cells) and myeloid cells (Mac-1+ or Gr+ positive cells) among BM cells, percentage of donor derived LT-HSCs, ST-HSCs and LMPPs in LSK cells and percentage of donor derived CLPs in lin-Sca1C-kit low cells in animals at post- transplantation (n=12). Error bars indicate mean±SD, ****P<0.0001, **P<0.01 and *P<0.05, Two-way ANOVA analysis and 2 tailed unpaired t-test.


( A ) of single LT - HSCs from septin 7fl / fl of septin 7 ∆ / ∆ mice that completed first second division up to 48 hours post initiation of culture . ( 192 sorted HSCs were analysed , error bars indicate mean ± SD ) .

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

(A) Percentage of single LT-HSCs from septin 7fl/fl of septin 7∆/∆ mice that completed first and second division up to 48 hours post initiation of culture. (192 sorted HSCs cells were analysed, error bars indicate mean±SD).


C . Western blot for POT1 DDR markers upon POT1 deletion induced with 0 . 5 μM 4 - OHT . Cl75 - parental . gRNA1 - POT1 knock - out in population by transient transfection with pSpCas9 ( BB ) - 2A - puro containing gRNA1 . EV - corresponding empty vector .

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

C. Western blot for POT1 and DDR markers upon POT1 deletion induced with 0.5 μM 4-OHT. Cl75 - parental cell line. gRNA1 - POT1 knock-out in the population by transient transfection with pSpCas9(BB)-2A-puro containing gRNA1 sequence. EV - corresponding empty vector.


D . Time course of telomere changes upon POT1 removal in clone 35 . d ( days ) TP0 - time point 0 , no 4OHT .

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

D. Time course of telomere length changes upon POT1 removal in clone 35. d (days) TP0 - time point 0, no 4OHT.


E . Growth curve for POT1 WT POT1 knockout . Population doublings ( PDL ) are represented as mean ± SD ( three biological replicates ) .

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

E. Growth curve for POT1 WT and POT1 knockout cells. Population doublings (PDL) are represented as mean ± SD (three biological replicates).


F . for TIFs in EdU positive EdU negative upon POT1 removal in clone 35 . bars show of containing more than 5 TIFs ± SD . Experiments were performed in triplicate . At least 120 were analyzed per condition per replicate . Significance was determined using two - way ANOVA . p - values are indicated on graph .

O O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O 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. Quantification for TIFs in EdU positive and EdU negative cells upon POT1 removal in clone 35. The bars show percentage of cells containing more than 5 TIFs ± SD. Experiments were performed in triplicate. At least 120 cells were analyzed per condition per replicate. Significance was determined using two-way ANOVA. p-values are indicated on the graph.


A . G - overhang for clone 35 upon POT1 removal . bars show relative amounts of single stranded telomeric ± SD . Experiments were performed in triplicate .

O O O O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O

A. G-overhang assay for clone 35 upon POT1 removal. The bars show relative amounts of single stranded telomeric DNA ± SD. Experiments were performed in triplicate.


B . 2D gels for telomeric from clone 35 with POT1 ( NT ) upon POT1 removal for 7 days ( 7d 4OHT ) .

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

B. 2D gels for telomeric DNA from clone 35 cells with POT1 (NT) and upon POT1 removal for 7 days (7d 4OHT).


C . Examples of metaphase spreads for non - treated ( NT ) clone 35 clone 35 treated with 4 - OHT for 4 7 days . arrowheads indicate fragile telomeres , red arrowhead indicates a sister - chromatid fusion . Scale bar equals 6 µm . D . of fragile telomeres ( left graph ) , sister chromatid fusions ( middle graph ) chromosome fusions ( right graph ) as a of events per metaphase . Experiments were performed in triplicate . At least 25 metaphases were examined per condition per replicate . mean is displayed error bars represent ± SD . Significance was determined using one - way ANOVA . p - values are indicated on graph .

O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR B-EXP_ASSAY 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 O O O O O O O O O O O O O O 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. Examples of metaphase spreads for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. White arrowheads indicate fragile telomeres, the red arrowhead indicates a sister-chromatid fusion. Scale bar equals 6 µm. D. Quantification of fragile telomeres (left graph), sister chromatid fusions (middle graph) and chromosome fusions (right graph) as a percentage of events per metaphase. Experiments were performed in triplicate. At least 25 metaphases were examined per condition per replicate. The mean is displayed and error bars represent ± SD. Significance was determined using one-way ANOVA. p-values are indicated on the graph.


E . representative of anaphases with bridges micronuclei for non - treated ( NT ) clone 35 clone 35 treated with 4 - OHT for 4 7 days . Scale bar equals 6 µm . Experiments were performed in triplicate . bars show of containing anaphases , anaphases with bridges or micronuclei + SD . At least 650 were examined per condition per replicate . Significance was determined using one - way ANOVA . p - values are indicated on graph . In experiments , POT1 deletion was induced with 0 . 5 μM 4 - OHT .

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

E. Quantification and representative images of anaphases with bridges and micronuclei for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Scale bar equals 6 µm. Experiments were performed in triplicate. The bars show percentage of cells containing anaphases, anaphases with bridges or micronuclei + SD. At least 650 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT.


A . Western blot for POT1 DDR markers upon POT1 removal in clone 35 overexpression of ectopic WT POT1 .

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

A. Western blot for POT1 and DDR markers upon POT1 removal in clone 35 and overexpression of ectopic WT POT1.


B . Telomere G - overhang for clone 35 upon POT1 removal overexpression of ectopic WT POT1 ( constant - field gel electrophoresis ) .

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

B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic WT POT1 (constant-field gel electrophoresis).


D , E Telomere G - overhang for clone 35 upon POT1 removal overexpression of ectopic POT1 variants carrying disease - associated mutations ( constant - field gel electrophoresis ) .

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

D, E Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic POT1 variants carrying disease-associated mutations (constant-field gel electrophoresis).


B . of precipitated telomeric in 2 - step QTIP ( % of input ) fold enrichment of precipitated telomeric compared with precipitated Alu repeat ( on dot blot analyses ) . Bars represent data from three independent experiments ± SD .

O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR 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

B. Quantification of precipitated telomeric DNA in 2-step QTIP (% of input) and fold enrichment of precipitated telomeric DNA compared with precipitated Alu repeat DNA (based on dot blot analyses). Bars represent data from three independent experiments ± SD.


C . Scatter plots representing immunoprecipitation specificity ( 2 - step QTIP / IgG ) versus difference between non treated ( NT ) at day 4 upon 4 - OHT induction ( 4d 4OHT / NT ) or difference between non treated ( NT ) at day 7 upon 4 - OHT induction ( 7d 4OHT / NT ) respectively ( data from Table EV1 ) . MRN in red , 9 - 1 - 1 in blue , shelterin in orange except for POT1 in black . Values are average of 3 biological replicates .

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

C. Scatter plots representing immunoprecipitation specificity (2-step QTIP/IgG ratio) versus difference between non treated cells (NT) and cells at day 4 upon 4-OHT induction (4d 4OHT/NT ratio) or difference between non treated cells (NT) and cells at day 7 upon 4-OHT induction (7d 4OHT/NT ratio) respectively (data from Table EV1). MRN complex in red, 9-1-1 complex in blue, shelterin complex in orange except for POT1 in black. Values are the average of the 3 biological replicates.


D . Heat map representing changes in of selected involved in damage ( MRN in red , 9 - 1 - 1 in blue ) HDR ( purple ) after POT1 knockout induction . marked with * pass t - , but not t - with Benjamini - Hochberg correction ( p < 0 . 05 , q < 0 . 09 ) . Data information : 4 - OHT - 4 - hydroxytamoxifen , NT - non - treated .

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

D. Heat map representing changes in levels of selected proteins involved in DNA damage (MRN complex in red, 9-1-1 complex in blue) and HDR proteins (purple) after POT1 knockout induction. Proteins marked with * pass t-test, but not t-test with Benjamini-Hochberg correction (p < 0.05, q < 0.09). Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells.


A . Telomere for clone 35 upon POT1 removal depletion of selected recombination with siRNA ( pulsed - field gel electrophoresis ) .

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

A. Telomere length for clone 35 upon POT1 removal and depletion of selected recombination proteins with siRNA (pulsed-field gel electrophoresis).


B . Telomere G - overhang for clone 35 upon POT1 removal inhibition of telomerase with 1 µM GRN163L ( pulsed - field gel electrophoresis ) .

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

B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and inhibition of telomerase with 1 µM GRN163L (pulsed-field gel electrophoresis).


C . DRIP for - hybrid detection at telomeres in clone 35 upon POT1 removal with S9 . 6 antibody . Dot blots represent two replicates . Bars represent data from three independent experiments ± SD . IgG signal was subtracted . Significance was determined using one - way ANOVA , p - values are indicated on graph .

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

C. DRIP assay for DNA-RNA hybrid detection at telomeres in clone 35 upon POT1 removal with S9.6 antibody. Dot blots represent two replicates. Bars represent data from three independent experiments ± SD. The IgG signal was subtracted. Significance was determined using one-way ANOVA, p-values are indicated on the graph.


D . C - circle ( CC ) for clone 35 upon POT1 removal . Bars represent data from three independent experiments ± SD . from ALT positive U2OS was used as positive control . Significance was determined using one - way ANOVA , p - values are indicated on graph . In experiments , POT1 deletions was induced with 0 . 5 μM 4 - OHT for 4 7 days .

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

D. C-circle (CC) assay for clone 35 upon POT1 removal. Bars represent data from three independent experiments ± SD. DNA from ALT positive cell line U2OS was used as positive control. Significance was determined using one-way ANOVA, p-values are indicated on the graph. In all experiments, POT1 deletions was induced with 0.5 μM 4-OHT for 4 and 7 days.


E . C - overhang for clone 35 upon POT1 removal ( pulsed - field gel electrophoresis , hybridization with Telo - G probe ) .

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

E. C-overhang length analysis for clone 35 upon POT1 removal (pulsed-field gel electrophoresis, hybridization with Telo-G probe).


F . representative pictures for visualization of PML ( IF , green ) telomeres ( FISH , red ) in non - treated ( NT ) clone 35 clone 35 treated with 4 - OHT for 4 7 days . Arrows indicate co - localizations . Scale bar equals 10 µm . Bars represent data from three independent experiments ± SD . At least 150 were examined per condition per replicate . Significance was determined using one - way ANOVA . p - values are indicated on graph . In experiments , POT1 deletion was induced with 0 . 5 μM 4 - OHT . Data information : 4 - OHT - 4 - hydroxytamoxifen , NT - non - treated , GRN163L - telomerase inhibitor Imitelstat , siNT - non - tagreting siRNA , negative control .

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

F. Quantification and representative pictures for visualization of the PML protein (IF, green) and telomeres (FISH, red) in non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Arrows indicate co-localizations. Scale bar equals 10 µm. Bars represent data from three independent experiments ± SD. At least 150 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT. Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells, GRN163L - telomerase inhibitor Imitelstat, siNT -non-tagreting siRNA, negative control.


( A ) RT - qPCR of relative miR - 21 - 3p miR - 21 - 5p in mouse brain , heart , kidney epidermis . N = 4 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 I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O

(A) RT-qPCR quantification of relative miR-21-3p and miR-21-5p levels in mouse brain, heart, kidney and epidermis. N=4 animals per group, one representative experiment is shown out of three independent replicates.


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

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 B-GENEPROD 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 of relative pri - miR - 21 miR - 21 - 3p in epidermis ( left middle ) , of relative miR - 21 - 3p in dermis ( right ) of acutely irradiated ( Ac - UV ; + ) non - irradiated ( - ) Ppard + / + - / - 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 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 B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE 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 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

(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 of relative miR - 21 - 3p in total skin of chronically irradiated ( Chr - UV ; + ) non - irradiated ( - ) Ppard + / + - / - 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 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 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 B-GENEPROD 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

(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 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O B-GENEPROD 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 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

(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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE 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 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE 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 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 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 I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE 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 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE 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 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O B-GENEPROD 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

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

(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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD 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 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 ) 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 B-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 B-ORGANISM 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 B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE 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

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

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

(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 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 B-GENEPROD 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 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 B-GENEPROD 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

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

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

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 .

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

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

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 .

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

O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 B-SUBCELLULAR B-GENEPROD 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 were treated with ATP8A1 siRNA1 for 72 h stained for indicated . Scale bars , 10 μm .

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

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

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 B-SUBCELLULAR O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 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 .

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

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 .

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

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 .

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

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 .

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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 ‐ 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 ) .

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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 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 .

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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 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 .

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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 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 .

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

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 O O O O O O O O O O O O O O B-TISSUE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O O

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

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

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

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 .

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

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 . .

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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 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 .

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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 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 .

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

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 .

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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 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 .

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