B . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , Y27632 ( 5 μM ) , and Ly294002 ( 15 μM ) treatment on 500 nM PGF2α - induced FOS gene expression in HRMECs ( n = 4 ) . C . Effect of KN93 ( 10 μM ) and RO - 318220 ( 250 nM ) treatment on 500 nM PGF2α - induced FOS expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test ( B , C Data are represented as mean ± SEM .

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B. Effect of U73122 (10 μM), BAPTA (50 μM), Y27632 (5 μM), and Ly294002 (15 μM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs (n=4). C. Effect of KN93 (10 μM) and RO-318220 (250 nM) treatment on 500 nM PGF2α-induced FOS expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, C Data are represented as mean ± SEM.


D . Effect of SB203580 ( 2 μM ) , PD98059 ( 20 μM ) , and SP600125 ( 60 nM ) treatment on 500 nM PGF2α - induced FOS gene expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test Data are represented as mean ± SEM .

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D. Effect of SB203580 (2 μM), PD98059 (20 μM), and SP600125 (60 nM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test Data are represented as mean ± SEM.


E . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , and KN93 ( 10 μM ) treatment on 500 nM PGF2α - induced p38 phosphorylation in HRMECs . F . Quantitation of the ratio of p38 phosphorylation to total p38 protein in E ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test ( F , Data are represented as mean ± SEM .

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E. Effect of U73122 (10 μM), BAPTA (50 μM), and KN93 (10 μM) treatment on 500 nM PGF2α-induced p38 phosphorylation in HRMECs. F. Quantitation of the ratio of p38 phosphorylation to total p38 protein in E (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test (F, Data are represented as mean ± SEM.


G . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , and SB203580 ( 2 μM ) on the phosphorylation of CAMK2 isoforms in 500 nM PGF2α - treated HRMECs . H . Quantitation of the ratio of CAMK2G phosphorylation to total CAMK2G in G ( n = 3 ) . I . Quantitation of the ratio of phosphorylated CAMK2D to total CAMK2D in G ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test H , I , Data are represented as mean ± SEM .

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G. Effect of U73122 (10 μM), BAPTA (50 μM), and SB203580 (2 μM) on the phosphorylation of CAMK2 isoforms in 500 nM PGF2α-treated HRMECs. H. Quantitation of the ratio of CAMK2G phosphorylation to total CAMK2G in G (n=3). I. Quantitation of the ratio of phosphorylated CAMK2D to total CAMK2D in G (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test H, I, Data are represented as mean ± SEM.


J . Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α - triggered p38 phosphorylation in HRMECs . K . Quantitation of the ratio of p38 phosphorylation to total p38 protein in J ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test Data are represented as mean ± SEM .

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J. Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α-triggered p38 phosphorylation in HRMECs. K. Quantitation of the ratio of p38 phosphorylation to total p38 protein in J (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test Data are represented as mean ± SEM.


L . Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α - induced FOS expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test L ) Data are represented as mean ± SEM .

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L. Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α-induced FOS expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test L) Data are represented as mean ± SEM.


M . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , KN93 ( 10 μM ) , and SB203580 ( 2 μM ) on ELK - 1 phosphorylation in 500 nM PGF2α - treated HRMECs . N . Quantitation of the ratio of phosphorylated ELK - 1 to total ELK - 1 in M ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test N ) . Data are represented as mean ± SEM .

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M. Effect of U73122 (10 μM), BAPTA (50 μM), KN93 (10 μM), and SB203580 (2 μM) on ELK-1 phosphorylation in 500 nM PGF2α-treated HRMECs. N. Quantitation of the ratio of phosphorylated ELK-1 to total ELK-1 in M (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test N). Data are represented as mean ± SEM.


B . Representative images of OIR retinas in WT and Cxcr2 - / - mice with or without AL8810 treatment . The green color shows the isolectin B4 - stained vessels , the second - row panels display the enlarged images of white boxes in the first - row panels , the third - row images show neovascular tufts ( NV , white ) , and the fourth - row images show the vaso - obliteration ( VO , white ) area . C . Quantitation of oxygen - induced retinal neovascularization in WT and Cxcr2 - / - mice with or without AL8810 treatment ( n = 12 ) . D . Quantitation of retinal vaso - obliteration in WT and Cxcr2 - / - mice with or without AL8810 treatment ( n = 12 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( C , D ) Scale bar : 500 μm ( unmagnified image ) , 150 μm ( magnified image ) . Data are represented as mean ± SEM .

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

B. Representative images of OIR retinas in WT and Cxcr2-/- mice with or without AL8810 treatment. The green color shows the isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in the first-row panels, the third-row images show neovascular tufts (NV, white), and the fourth-row images show the vaso-obliteration (VO, white) area. C. Quantitation of oxygen-induced retinal neovascularization in WT and Cxcr2-/- mice with or without AL8810 treatment (n=12). D. Quantitation of retinal vaso-obliteration in WT and Cxcr2-/- mice with or without AL8810 treatment (n=12). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (C, D) Scale bar: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.


E . Effect of AL8810 on retinal Fos expression in WT and Cxcr2 - / - OIR mice on postnatal day 16 ( n = 6 ) . F . Effect of AL8810 on retinal Cxcl1 expression in WT and Cxcr2 - / - OIR mice on postnatal day 16 ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Mann - Whitney test ( E , F ) . Data are represented as mean ± SEM .

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E. Effect of AL8810 on retinal Fos expression in WT and Cxcr2-/- OIR mice on postnatal day 16 (n=6). F. Effect of AL8810 on retinal Cxcl1 expression in WT and Cxcr2-/- OIR mice on postnatal day 16 (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Mann-Whitney test (E, F). Data are represented as mean ± SEM.


( D , E ) Representative immunofluorescence microscopy images of the distribution of septin 7 ( red ) or borg3 or borg4 ( green ) in young , aged and aged LT - HSCs treated with CASIN ( 5µM ) . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm .

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(D,E) Representative immunofluorescence microscopy images of the distribution of septin 7 (red) or borg3 or borg4 (green) in young, aged and aged LT-HSCs treated with CASIN (5µM). Nuclei were stained with DAPI (blue), scale bar= 5µm.


( A ) Representative immunofluorescence confocal microscopy images of young , aged and aged + CASIN ( 5µM ) treated LT - HSCs for the proximity ligation assay ( red signal ) , testing the extent of close physical interaction of Cdc42 and borg4 . A red fluorescent signal indicates close proximity of the two proteins tested . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm . ( B ) Quantification of the level of fluorescent signal in young , aged and aged + CASIN ( 5µM ) treated LT - HSCs to quantify the Cdc42 - borg4 proximity interaction . Data are normalized to the mean fluorescence signal of aged LT - HSCs . 3 biological repeats , n = at least 17 cells per condition , error bars indicate mean ± SEM , unpaired t - test , * * * * P < 0 . 0001 . ( C ) Representative immunofluorescence confocal microscopy images of young , aged and aged + CASIN ( 5µM ) treated LT - HSCs for the proximity ligation assay ( red signal ) , testing the extent of close physical interaction of borg4 and septin 7 . A red fluorescent signal indicates close proximity of the two proteins tested . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm . ( D ) Quantification of the level of fluorescent signal in young , aged and aged + CASIN ( 5µM ) treated LT - HSCs to quantify the borg4 - septin 7 proximity interaction . Data are normalized to the mean fluorescence signal of aged LT - HSCs . 3 biological repeats , n = at least 17 cells per condition , error bars indicate mean ± SEM , unpaired t - test , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(A) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of Cdc42 and borg4. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (B) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the Cdc42-borg4 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ****P<0.0001. (C) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of borg4 and septin 7. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (D) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the borg4-septin 7 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ***P<0.001, ****P<0.0001.


( 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 .

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(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 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 .

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(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 ) 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 .

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(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 ) 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 ) .

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(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 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 .

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

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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 and POT1 knockout cells . Population doublings ( PDL ) are represented as mean ± SD ( three biological replicates ) .

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


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 .

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

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

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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 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 .

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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 . 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 .

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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 and DDR markers upon POT1 removal in clone 35 and overexpression of ectopic WT POT1 .

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


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

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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 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 ) .

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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 . 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 .

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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 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 .

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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 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 .

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

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


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 ) .

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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 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 .

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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 ) 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 .

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

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


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 .

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

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(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 ) 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 :

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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