F Changes in DNA methylation at nephrin gene promoter by GSK - J4 ( 40 μM ) in immortalized podocytes . U , unmethylated - specific primers ; M , methylated - specific primers .

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F Changes in DNA methylation at nephrin gene promoter by GSK-J4 (40 μM) in immortalized podocytes. U, unmethylated-specific primers; M, methylated-specific primers.


Relative mRNA levels of KDM6A , nephrin and WT - 1 , normalized to β - actin , expressed in kidney glomeruli of normal and diabetic mice at 4 , 8 and 12 weeks after diabetic induction . * Significant differences ( P < 0 . 05 ) compared with normal controls ( Wilcoxon two - sample test ; n = 8 each ) .

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Relative mRNA levels of KDM6A, nephrin and WT-1, normalized to β-actin, expressed in kidney glomeruli of normal and diabetic mice at 4, 8 and 12 weeks after diabetic induction. *Significant differences (P < 0.05) compared with normal controls (Wilcoxon two-sample test; n =8 each).


B Western blot analysis of nephrin , KDM6A and H3K27me3 in kidney glomeruli of normal and diabetic mice at different time points . * P < 0 . 05 versus normal controls for the indicated time points ( Wilcoxon two - sample test ; n = 3 ) .

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B Western blot analysis of nephrin, KDM6A and H3K27me3 in kidney glomeruli of normal and diabetic mice at different time points. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3).


C Changes in body weights and levels of blood glucose in normal , diabetic and GSK - J4 - treated diabetic mice . As noted , GSK - J4 treatment did not significantly affect body weights or blood glucose levels in diabetic mice during the experimental period of 12 weeks ( Wilcoxon two - sample test ; n = 8 ) .

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C Changes in body weights and levels of blood glucose in normal, diabetic and GSK-J4-treated diabetic mice. As noted, GSK-J4 treatment did not significantly affect body weights or blood glucose levels in diabetic mice during the experimental period of 12 weeks (Wilcoxon two-sample test; n = 8).


D Levels of urinary protein excretion , weights of kidney , and levels of HbA1c in normal , diabetic and GSK - J4 - treated diabetic mice . Urinary total protein excretion , kidney weight and levels of HbA1c were measured at 12 weeks after diabetic induction . The mean relative kidney weight ( % ) shown in the study is determined as the percent of kidneys out of total body weight , and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin ( % HbA1c ; DCCT unit ) . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated diabetic mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 8 ) .

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D Levels of urinary protein excretion, weights of kidney, and levels of HbA1c in normal, diabetic and GSK-J4-treated diabetic mice. Urinary total protein excretion, kidney weight and levels of HbA1c were measured at 12 weeks after diabetic induction. The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8).


E Western blot analysis of KDM6A , nephrin , WT - 1 and H3K27me3 expressed in kidney glomeruli of normal , diabetic and GSK - J4 - treated diabetic mice at 12 weeks after diabetic induction . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated diabetic mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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E Western blot analysis of KDM6A, nephrin, WT-1 and H3K27me3 expressed in kidney glomeruli of normal, diabetic and GSK-J4-treated diabetic mice at 12 weeks after diabetic induction. *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


F Immunofluorescence analysis of KDM6A , nephrin and WT - 1 in kidney sections from normal , diabetic , and GSK - J4 - treated diabetic mice . Green : nephrin or WT - 1 ; Red : KDM6A ; Blue : DAPI . Scale bars , 20 μm . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated diabetic mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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F Immunofluorescence analysis of KDM6A, nephrin and WT-1 in kidney sections from normal, diabetic, and GSK-J4-treated diabetic mice. Green: nephrin or WT-1; Red: KDM6A; Blue: DAPI. Scale bars, 20 μm. *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


G Immunofluorescence staining of F - actin and KDM6A in primary podocytes isolated from normal , diabetic , and GST - J4 - treated diabetic mice . Green : F - actin ; Red : KDM6A ; Blue : DAPI . Scale bars , 20 μm . Presented experiments were performed at least three times independently .

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G Immunofluorescence staining of F-actin and KDM6A in primary podocytes isolated from normal, diabetic, and GST-J4-treated diabetic mice. Green: F-actin; Red: KDM6A; Blue: DAPI. Scale bars, 20 μm. Presented experiments were performed at least three times independently.


A Quantitative RT - PCR evaluation of KDM6A mRNA expression in kidney glomeruli isolated from wild - type and KDM6A - knockout ( KO ) mice . * P < 0 . 05 , significant difference versus wild - type controls ( Wilcoxon two - sample test ; n = 8 ) .

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A Quantitative RT-PCR evaluation of KDM6A mRNA expression in kidney glomeruli isolated from wild-type and KDM6A-knockout (KO) mice. *P < 0.05, significant difference versus wild-type controls (Wilcoxon two-sample test; n = 8).


B Western blot analysis of KDM6A expression in glomeruli and podocytes isolated from wild - type and KDM6A - KO mice . Presented experiments were performed at least three times independently .

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B Western blot analysis of KDM6A expression in glomeruli and podocytes isolated from wild-type and KDM6A-KO mice. Presented experiments were performed at least three times independently.


C Changes in body weights and levels of blood glucose in wild - type or KDM6A - KO mice with or without STZ treatment . No significant differences in body weights or blood glucose levels between wild - type and KDM6A - KO mice with diabetes were observed during the 8 - week experimental period ( Wilcoxon two - sample test ; n = 8 ) .

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C Changes in body weights and levels of blood glucose in wild-type or KDM6A-KO mice with or without STZ treatment. No significant differences in body weights or blood glucose levels between wild-type and KDM6A-KO mice with diabetes were observed during the 8-week experimental period (Wilcoxon two-sample test; n = 8).


D Levels of urinary protein excretion , weights of kidney and levels of HbA1c in wild - type and KDM6A - KO mice with or without STZ treatment ( at 8 weeks after the onset of diabetes ) . The mean relative kidney weight ( % ) shown in the study is determined as the percent of kidneys out of total body weight , and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin ( % HbA1c ; DCCT unit ) . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 8 ) .

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D Levels of urinary protein excretion, weights of kidney and levels of HbA1c in wild-type and KDM6A-KO mice with or without STZ treatment (at 8 weeks after the onset of diabetes). The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8).


E Immunofluorescence images of kidney sections stained with KDM6A and nephrin in wild - type ( WT - NC ) , KDM6A - KO ( KO - NC ) , STZ - treated wild - type ( WT - DM ) , or STZ - treated KDM6A - KO ( KO - DM ) mice . Scale bars , 20 μm . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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E Immunofluorescence images of kidney sections stained with KDM6A and nephrin in wild-type (WT-NC), KDM6A-KO (KO-NC), STZ-treated wild-type (WT-DM), or STZ-treated KDM6A-KO (KO-DM) mice. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


F Immunofluorescence images of kidney sections stained with KDM6A and H3K27me3 in wild - type ( WT - NC ) , KDM6A - KO ( KO - NC ) , STZ - treated wild - type ( WT - DM ) , or STZ - treated KDM6A - KO ( KO - DM ) mice . Scale bars , 20 μm . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

F Immunofluorescence images of kidney sections stained with KDM6A and H3K27me3 in wild-type (WT-NC), KDM6A-KO (KO-NC), STZ-treated wild-type (WT-DM), or STZ-treated KDM6A-KO (KO-DM) mice. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


G Immunofluorescence staining of F - actin and KDM6A in primary podocytes isolated from the above treated mice . Presented experiments were performed at least three times independently . Scale bars , 20 μm .

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G Immunofluorescence staining of F-actin and KDM6A in primary podocytes isolated from the above treated mice. Presented experiments were performed at least three times independently. Scale bars, 20 μm.


H Western blot analysis of nephrin , KDM6A and WT - 1 expressed in primary podocytes isolated from the above treated mice . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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H Western blot analysis of nephrin, KDM6A and WT-1 expressed in primary podocytes isolated from the above treated mice. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


I Effect of high glucose on the expression of podocyte - related markers in primary cultured podocytes isolated from wild - type or KDM6A - KO mice . The primary podocytes isolated from wild - type or KDM6A - KO mice were cultured in normal or high glucose ( 30 mM ) for 48 hours . Protein lysates from the cultured podocytes were subjected to Western blot analysis with the indicated antibodies . * P < 0 . 05 versus wild - type podocytes in normal glucose , # P < 0 . 05 versus wild - type podocytes in high glucose ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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I Effect of high glucose on the expression of podocyte-related markers in primary cultured podocytes isolated from wild-type or KDM6A-KO mice. The primary podocytes isolated from wild-type or KDM6A-KO mice were cultured in normal or high glucose (30 mM) for 48 hours. Protein lysates from the cultured podocytes were subjected to Western blot analysis with the indicated antibodies. *P < 0.05 versus wild-type podocytes in normal glucose, #P < 0.05 versus wild-type podocytes in high glucose (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


A Screening of the potential KDM6A - regulated transcriptional factors involved in repression of nephrin expression . After transduction with a control lentiviral vector or a lentiviral vector expressing KDM6A into primary podocytes for 48 hours , intracellular RNAs were isolated and used for RNA sequencing ( RNA - Seq ) analysis . The transcript expression patterns of 86 selected transcriptional factors from three independent RNA - Seq experiments are presented in a heat map . Notably , among these 86 selected genes , KLF10 is the most upregulated gene .

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A Screening of the potential KDM6A-regulated transcriptional factors involved in repression of nephrin expression. After transduction with a control lentiviral vector or a lentiviral vector expressing KDM6A into primary podocytes for 48 hours, intracellular RNAs were isolated and used for RNA sequencing (RNA-Seq) analysis. The transcript expression patterns of 86 selected transcriptional factors from three independent RNA-Seq experiments are presented in a heat map. Notably, among these 86 selected genes, KLF10 is the most upregulated gene.


B Validation of increased KDM6A and KLF10 expression in primary podocytes that were infected with an empty lentiviral vector or KDM6A - expressing lentiviral vector for 48 hours . * P < 0 . 05 versus the empty vector control ( Wilcoxon two - sample test ; n = 3 ) .

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B Validation of increased KDM6A and KLF10 expression in primary podocytes that were infected with an empty lentiviral vector or KDM6A-expressing lentiviral vector for 48 hours. *P < 0.05 versus the empty vector control (Wilcoxon two-sample test; n = 3).


C Increased expression of KDM6A and KLF10 in primary podocytes cultured in high glucose for 48 hours . * P < 0 . 05 versus normal controls ( Wilcoxon two - sample test ; n = 3 ) .

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C Increased expression of KDM6A and KLF10 in primary podocytes cultured in high glucose for 48 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).


D Effect of KLF10 knockdown on high glucose - mediated reduction of nephrin in primary podocytes . As noted , knockdown of KLF10 prevented downregulation of nephrin and upregulation of KDM6A in high glucose - treated podocytes . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus control siRNA with HG incubation ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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D Effect of KLF10 knockdown on high glucose-mediated reduction of nephrin in primary podocytes. As noted, knockdown of KLF10 prevented downregulation of nephrin and upregulation of KDM6A in high glucose-treated podocytes. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


E Immunofluorescence analysis of KLF10 and nephrin in renal sections of normal , diabetic , and GSK - J4 - treated diabetic mice . Scale bars , 20 μm . * P < 0 . 05 versus the normal control group , # P < 0 . 05 versus the untreated diabetic group ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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E Immunofluorescence analysis of KLF10 and nephrin in renal sections of normal, diabetic, and GSK-J4-treated diabetic mice. Scale bars, 20 μm. *P < 0.05 versus the normal control group, #P < 0.05 versus the untreated diabetic group (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


F Immunofluorescence images of KLF10 and nephrin in renal sections of wild - type or KDM6A - KO mice with or without STZ treatment . Scale bars , 20 μm . * P < 0 . 05 versus the untreated wild - type group , # P < 0 . 05 versus the STZ - treated wild - type group ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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F Immunofluorescence images of KLF10 and nephrin in renal sections of wild-type or KDM6A-KO mice with or without STZ treatment. Scale bars, 20 μm. *P < 0.05 versus the untreated wild-type group, #P < 0.05 versus the STZ-treated wild-type group (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


G Western blot analysis of KDM6A , KFL10 and nephrin expression in primary podocytes isolated from normal , diabetic , and GSK - J4 - treated diabetic mice . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus the untreated diabetic group ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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G Western blot analysis of KDM6A, KFL10 and nephrin expression in primary podocytes isolated from normal, diabetic, and GSK-J4-treated diabetic mice. *P < 0.05 versus normal controls, #P < 0.05 versus the untreated diabetic group (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


H Western blot analysis of KDM6A , KLF10 and nephrin expression in primary podocytes isolated from wild - type or KDM6A - KO mice with or without STZ treatment . * P < 0 . 05 versus the untreated wild - type group , # P < 0 . 05 versus the STZ - treated wild - type group ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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H Western blot analysis of KDM6A, KLF10 and nephrin expression in primary podocytes isolated from wild-type or KDM6A-KO mice with or without STZ treatment. *P < 0.05 versus the untreated wild-type group, #P < 0.05 versus the STZ-treated wild-type group (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


ChIP analysis of KLF10 , acetyl - Histone H4 ( H4 - Ac ) , Dnmt1 and Dnmt3 binding to nephrin gene promoter . ChIP assays were carried out using cross - linked chromatin from primary podocytes that were cultured in normal or high glucose conditions . * P < 0 . 05 , significant difference versus the normal control group ( Wilcoxon two - sample test ; n = 3 ) .

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ChIP analysis of KLF10, acetyl-Histone H4 (H4-Ac), Dnmt1 and Dnmt3 binding to nephrin gene promoter. ChIP assays were carried out using cross-linked chromatin from primary podocytes that were cultured in normal or high glucose conditions. *P < 0.05, significant difference versus the normal control group (Wilcoxon two-sample test; n = 3).


J Modulation of podocyte - specific marker expression in primary podocytes by KLF10 overexpression . Ectopic overexpression of KLF10 in primary podocytes significantly repressed various podocyte - specific markers , but conversely increased KDM6A expression . * P < 0 . 05 versus the empty vector control ( Wilcoxon two - sample test ; n = 3 ) .

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J Modulation of podocyte-specific marker expression in primary podocytes by KLF10 overexpression. Ectopic overexpression of KLF10 in primary podocytes significantly repressed various podocyte-specific markers, but conversely increased KDM6A expression. *P < 0.05 versus the empty vector control (Wilcoxon two-sample test; n = 3).


A Quantitative RT - PCR evaluation of KLF10 mRNA expression in kidney samples of wild - type and KLF10 - knockout ( KO ) mice . * * P < 0 . 0001 , significant difference versus wild - type controls ( Wilcoxon two - sample test ; n = 8 ) .

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A Quantitative RT-PCR evaluation of KLF10 mRNA expression in kidney samples of wild-type and KLF10-knockout (KO) mice. **P < 0.0001, significant difference versus wild-type controls (Wilcoxon two-sample test; n = 8).


B Western blot analysis of KLF10 expression in kidney samples of wild - type or KLF10 - KO mice treated with or without STZ at 8 weeks after the onset of diabetes .

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B Western blot analysis of KLF10 expression in kidney samples of wild-type or KLF10-KO mice treated with or without STZ at 8 weeks after the onset of diabetes.


C Changes in body weight and blood glucose levels in wild - type or KLF10 - KO mice with or without STZ treatment . No significant differences in body weights or blood glucose levels between diabetic wild - type mice and diabetic KLF10 - KO mice were detected ( Wilcoxon two - sample test ; n = 8 ) .

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C Changes in body weight and blood glucose levels in wild-type or KLF10-KO mice with or without STZ treatment. No significant differences in body weights or blood glucose levels between diabetic wild-type mice and diabetic KLF10-KO mice were detected (Wilcoxon two-sample test; n = 8).


D Levels of urinary protein excretion , weights of kidney and levels of HbA1c in wild - type or KLF10 - KO mice with or without STZ treatment . The mean relative kidney weight ( % ) shown in the study is determined as the percent of kidneys out of total body weight , and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin ( % HbA1c ; DCCT unit ) . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 8 ) .

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D Levels of urinary protein excretion, weights of kidney and levels of HbA1c in wild-type or KLF10-KO mice with or without STZ treatment. The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8).


E Immunofluorescence images of kidney sections stained with KLF10 and nephrin in wild - type or KLF10 - KO mice untreated or treated with STZ . Scale bars , 20 μm . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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E Immunofluorescence images of kidney sections stained with KLF10 and nephrin in wild-type or KLF10-KO mice untreated or treated with STZ. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


F Immunofluorescence images of kidney sections stained with KLF10 and KDM6A in wild - type or KLF10 - KO mice untreated or treated with STZ . Scale bars , 20 μm . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

F Immunofluorescence images of kidney sections stained with KLF10 and KDM6A in wild-type or KLF10-KO mice untreated or treated with STZ. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


G Immunofluorescence staining of F - actin and KLF10 in primary podocytes isolated from the wild - type or KLF10 - KO mice untreated or treated with STZ . Presented experiments were performed at least three times independently . Scale bars , 20 μm .

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G Immunofluorescence staining of F-actin and KLF10 in primary podocytes isolated from the wild-type or KLF10-KO mice untreated or treated with STZ. Presented experiments were performed at least three times independently. Scale bars, 20 μm.


H Immunofluorescence staining of F - actin and KDM6A in primary podocytes isolated from the wild - type or KLF10 - KO mice untreated or treated with STZ . Presented experiments were performed at least three times independently . Scale bars , 20 μm .

H Immunofluorescence staining of F-actin and KDM6A in primary podocytes isolated from the wild-type or KLF10-KO mice untreated or treated with STZ. Presented experiments were performed at least three times independently. Scale bars, 20 μm.


I Western blot analysis of KLF10 , KDM6A , nephrin and WT - 1 expression in primary podocytes isolated from the wild - type or KLF10 - KO mice that were untreated or treated with STZ . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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I Western blot analysis of KLF10, KDM6A, nephrin and WT-1 expression in primary podocytes isolated from the wild-type or KLF10-KO mice that were untreated or treated with STZ. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


J Western blot analysis of KLF10 , KDM6A , nephrin and WT - 1 expressed in podocytes treated with TGF - β1 or the combination of TGF - β1 and KLF10 knockdown . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus control siRNA with TGF - β1 treatment ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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J Western blot analysis of KLF10, KDM6A, nephrin and WT-1 expressed in podocytes treated with TGF-β1 or the combination of TGF-β1 and KLF10 knockdown. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with TGF-β1 treatment (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


A Immunofluorescence analysis of kidney sections from diabetic nephropathy subjects and non - diabetic controls stained with KDM6A , KLF10 , nephrin and WT - 1 . Scale bars , 50 μm . * P < 0 . 05 by Wilcoxon two - sample test ( n = 6 for each group ) .

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A Immunofluorescence analysis of kidney sections from diabetic nephropathy subjects and non-diabetic controls stained with KDM6A, KLF10, nephrin and WT-1. Scale bars, 50 μm. *P < 0.05 by Wilcoxon two-sample test (n = 6 for each group).


B Western blot analysis of kidney tissues from non - diabetic controls and diabetic nephropathy subjects . Expression levels of KDM6A , KLF10 , nephrin and WT - 1 in kidney tissues were determined by immunoblotting using the indicated antibodies . Relative protein levels in kidney tissues were normalized to actin . * P < 0 . 05 by Wilcoxon two - sample test ( n = 6 for each group ) .

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B Western blot analysis of kidney tissues from non-diabetic controls and diabetic nephropathy subjects. Expression levels of KDM6A, KLF10, nephrin and WT-1 in kidney tissues were determined by immunoblotting using the indicated antibodies. Relative protein levels in kidney tissues were normalized to actin. *P < 0.05 by Wilcoxon two-sample test (n = 6 for each group).


C Levels of KDM6A and KLF10 , nephrin mRNAs in urine exosomes of diabetic nephropathy patients and non - diabetic controls . Relative mRNA levels in human urinary exosomes were normalized to 18S rRNA . Horizontal lines are medians . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 by Wilcoxon two - sample test ( n = 12 for each group ) .

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C Levels of KDM6A and KLF10, nephrin mRNAs in urine exosomes of diabetic nephropathy patients and non-diabetic controls. Relative mRNA levels in human urinary exosomes were normalized to 18S rRNA. Horizontal lines are medians. *P < 0.05, **P < 0.01, and ***P < 0.001 by Wilcoxon two-sample test (n = 12 for each group).


Micrographs of maximum intensity projections of forebrain sections from 3 mo old GLASTCreERT2 / eGFP mice 21 days after tamoxifen treatment Double - positive cells are indicated by arrowheads . CTX : cerebral cortex ; DIE : diencephalon ; HC : hippocampus . Scale bars : 300 µm ( A ) , 20 µm ( B ) , 75 µm ( C ) , 30 µm ( D ) , Histograms showing quantifications ( B , n = 6 animals ; 5 region of interest ( ROI ) covering the diencephalon on a total of 3 slides analyzed per animal Data information : data are presented as mean ± SEM . Each data point represents one animal .

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Micrographs of maximum intensity projections of forebrain sections from 3 mo old GLASTCreERT2/eGFP mice 21 days after tamoxifen treatment Double-positive cells are indicated by arrowheads. CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 300 µm (A), 20 µm (B), 75 µm (C), 30 µm (D), Histograms showing quantifications (B, n = 6 animals; 5 region of interest (ROI) covering the diencephalon on a total of 3 slides analyzed per animal Data information: data are presented as mean ± SEM. Each data point represents one animal.


Micrographs of maximum intensity projections of forebrain sections from 3 mo old C57BL / 6J mice after EdU application for 4 weeks in drinking water ( F ) immunostained as indicated on top of the panels . Double - positive cells are indicated by arrowheads . CTX : cerebral cortex ; DIE : diencephalon ; HC : hippocampus . Scale bars : 10 µm ( F ) .

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Micrographs of maximum intensity projections of forebrain sections from 3 mo old C57BL/6J mice after EdU application for 4 weeks in drinking water (F) immunostained as indicated on top of the panels. Double-positive cells are indicated by arrowheads. CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 10 µm (F).


G ) Histograms showing quantifications for n = 5 animals ; 5 ROI covering the diencephalon on a total of 2 slides analyzed per animal ) . Data information : data are presented as mean ± SEM . Each data point represents one animal .

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G) Histograms showing quantifications for n = 5 animals; 5 ROI covering the diencephalon on a total of 2 slides analyzed per animal). Data information: data are presented as mean ± SEM. Each data point represents one animal.


A - E Micrographs of maximum intensity projections ( A - D ) or single optical section ( E ) of forebrain sections stained as indicated for fluorescent proteins in astrocytes labelled in 8 mo ( A , B , E ) and 3 mo ( C , D ) GLASTCreERT2 / Confetti mice at 21 dpt . Note that clusters of duplets ( D ) and triplets ( C ) labelled in the same color can be found only in the DIE . Also note that astrocyte somata are often very close together , as also observed after live in vivo imaging of dividing astrocytes Somata of single cells are highlighted in circles . Data information : CTX : cerebral cortex ; DIE : diencephalon ; HC : hippocampus . Scale bars : 300 µm ( A ) , 100 µm ( B ) , 50 µm ( C , D ) , 20 µm ( E ) .

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A-E Micrographs of maximum intensity projections (A - D) or single optical section (E) of forebrain sections stained as indicated for fluorescent proteins in astrocytes labelled in 8 mo (A, B, E) and 3 mo (C, D) GLASTCreERT2/Confetti mice at 21 dpt. Note that clusters of duplets (D) and triplets (C) labelled in the same color can be found only in the DIE. Also note that astrocyte somata are often very close together, as also observed after live in vivo imaging of dividing astrocytes Somata of single cells are highlighted in circles. Data information: CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 300 µm (A), 100 µm (B), 50 µm (C, D), 20 µm (E).


B Micrographs of forebrain sections of 3 mo old GLASTCreERT2 / RPL22HA / eGFP 21 dpt mice stained for HA show maximum intensity projections of HA + expressing cells with astroglial morphology . CTX GM : cerebral cortex grey matter ; DIE : diencephalon . Scale bars : 100 µm . C Micrographs of forebrain sections of 3 mo old GLASTCreERT2 / RPL22HA / eGFP mice 21dpt stained for HA and S100β showing the specificity of the mouse line with HA detected only in S100β + astrocytes . DIE : diencephalon . Scale bar : 70 μm . Data information : CTX GM : cerebral cortex grey matter ; DIE : diencephalon ; n = 3 biological replicates , tissue from 3 mice was

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Micrographs of forebrain sections of 3 mo old GLASTCreERT2/RPL22HA/eGFP 21 dpt mice stained for HA show maximum intensity projections of HA+ expressing cells with astroglial morphology. CTX GM: cerebral cortex grey matter; DIE: diencephalon. Scale bars: 100 µm. C Micrographs of forebrain sections of 3 mo old GLASTCreERT2/RPL22HA/eGFP mice 21dpt stained for HA and S100β showing the specificity of the mouse line with HA detected only in S100β+ astrocytes. DIE: diencephalon. Scale bar: 70 μm. Data information: CTX GM: cerebral cortex grey matter; DIE: diencephalon; n=3 biological replicates, tissue from 3 mice was pooled


D Histogram showing expression levels of the top 10 astrocyte specific markers genes in DIE ( blue bars ) and CTX GM ( red bars ) samples . Data information : CTX GM : cerebral cortex grey matter ; DIE : diencephalon ; n = 3 biological replicates , tissue from 3 mice was

D Histogram showing expression levels of the top 10 astrocyte specific markers genes in DIE (blue bars) and CTX GM (red bars) samples. Data information: CTX GM: cerebral cortex grey matter; DIE: diencephalon; n=3 biological replicates, tissue from 3 mice was pooled


C Example of a Smad4 / GFP double - positive astrocyte ( indicated by arrowhead ) in a single optical section of DIE from 3 mo old GLASTCreERT2 / eGFP mice 21 dpt immunostained as indicated on top of the panel . Orthogonal projections of the area highlighted in left panel of C shown at higher magnification in the right panel . Scale bar : 15 µm .

C Example of a Smad4/GFP double-positive astrocyte (indicated by arrowhead) in a single optical section of DIE from 3 mo old GLASTCreERT2/eGFP mice 21 dpt immunostained as indicated on top of the panel. Orthogonal projections of the area highlighted in left panel of C shown at higher magnification in the right panel. Scale bar: 15 µm.


D Example of a proliferating ( PCNA + , arrowhead ) astrocyte in a single optical section of DIE from 3 mo old GLASTCreERT2 / eGFP mice 21 dpt immunostained as indicated on top with single channels shown in middle and right panels . Scale bars : 15 µm .

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D Example of a proliferating (PCNA+, arrowhead) astrocyte in a single optical section of DIE from 3 mo old GLASTCreERT2/eGFP mice 21 dpt immunostained as indicated on top with single channels shown in middle and right panels. Scale bars: 15 µm.


E Histogram depicting percentage of PCNA positive cells of all GFP positive cells in the DIE of the genotype indicated on the x - axis ( n = 6 animals per genotype ; 5 ROI covering the diencephalon on a total of 3 slides were analyzed per animal ; PCNA negative and positive cells comparing wt / wt vs . fl / fl animals , adjusted P - value = 0 . 002 ) . Data information : data are presented as mean ± SEM . Each dot represents one n . * P < 0 . 05 , * * P < 0 . 01 nonparametric 2way ANOVA followed by Sidak ' s multiple comparisons

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E Histogram depicting percentage of PCNA positive cells of all GFP positive cells in the DIE of the genotype indicated on the x-axis (n = 6 animals per genotype; 5 ROI covering the diencephalon on a total of 3 slides were analyzed per animal; PCNA negative and positive cells comparing wt/wt vs. fl/fl animals, adjusted P-value = 0.002). Data information: data are presented as mean ± SEM. Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric 2way ANOVA followed by Sidak's multiple comparisons test


F , G Histograms depicting percentage of the neurosphere - generating cells obtained from the DIE ( F ) or SEZ ( G ) of GLASTCreERT2 / Smad4fl / fl / eGFP or GLASTCreERT2 / Smad4WT / WT / eGFP , as indicated on the x - axis ( n = 3 - 4 animals for SEZ , 4 to 8 technical replicates analyzed per animal ; 4 animals for DIE , 8 technical replicates analyzed per animal ) . Data information : data are presented as mean ± SEM . F : GFP negative and GFP positive cells comparing wt / wt vs fl / fl animals , adjusted P - value = 0 . 0408 ; G : GFP negative and GFP positive cells comparing wt / wt vs fl / fl animals , ns ) . Each dot represents one n . * P < 0 . 05 , * * P < 0 . 01 nonparametric 2way ANOVA followed by Sidak ' s multiple comparisons

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F, G Histograms depicting percentage of the neurosphere-generating cells obtained from the DIE (F) or SEZ (G) of GLASTCreERT2/Smad4fl/fl/eGFP or GLASTCreERT2/Smad4WT/WT/eGFP, as indicated on the x-axis (n = 3-4 animals for SEZ, 4 to 8 technical replicates analyzed per animal; 4 animals for DIE, 8 technical replicates analyzed per animal). Data information: data are presented as mean ± SEM. F: GFP negative and GFP positive cells comparing wt/wt vs fl/fl animals, adjusted P-value = 0.0408; G: GFP negative and GFP positive cells comparing wt/wt vs fl/fl animals, ns). Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric 2way ANOVA followed by Sidak's multiple comparisons test


H Histogram depicting the number of secondary neurospheres generated from 10000 Smad4f / f neurosphere cells at 7 days after infection with MLV - based retrovirus containing either CAG - NLS - Cre or only CAG - GFP ( n = 11 cultures / animals , 3 technical replicates per condition analyzed ; control vs . Cre , P - value = 0 . 0149 ) . Note the decrease in DIE astrocyte proliferation in vivo and in neurosphere formation upon Smad4 deletion Data information : data are presented as mean ± SEM . Each dot represents one n . * P < 0 . 05 , * * P < 0 . 01 nonparametric Mann - Whitney test ) .

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H Histogram depicting the number of secondary neurospheres generated from 10000 Smad4f/f neurosphere cells at 7 days after infection with MLV-based retrovirus containing either CAG-NLS-Cre or only CAG-GFP (n = 11 cultures / animals, 3 technical replicates per condition analyzed; control vs. Cre, P-value = 0.0149). Note the decrease in DIE astrocyte proliferation in vivo and in neurosphere formation upon Smad4 deletion Data information: data are presented as mean ± SEM. Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric Mann-Whitney test).


B Immunofluorescence analysis of fibronectin ( green ) and collagen ( red ) fibers on de - cellularized ECM produced by human fibroblasts . Fibers orientation was quantified using ImageJ software . Percentages indicate oriented fibers accumulated in a range of ± 21 ° around the modal angle . Data is represented as mean ± s . d . ( n = 10 random fields from 2 independent determinations ) . . Scale bar , 50 µm . A representative image of picrosirius red staining from 10 analyzed is shown for each condition .

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B Immunofluorescence analysis of fibronectin (green) and collagen (red) fibers on de-cellularized ECM produced by human fibroblasts. Fibers orientation was quantified using ImageJ software. Percentages indicate oriented fibers accumulated in a range of ± 21° around the modal angle. Data is represented as mean ± s.d. (n=10 random fields from 2 independent determinations)..Scale bar, 50 µm. A representative image of picrosirius red staining from 10 analyzed is shown for each condition.


D , E Quantification of proliferation of 1205Lu ( D ) and SKMEL5 ( E ) cells plated for 48 h on plastic , Coll - 1 or the indicated fibroblast - derived ECMs prior a 96 h treatment with vehicle , 5 µM BRAFi or 2 µM BRAFi plus 0 . 01 µM MEKi . Cells were counted by Hoechst - labeled nuclei staining . Data are represented as bar plots with mean ± SEM normalized to vehicle of 3 independent experiments . ( D ) * * P = 0 . 015 , * * * P = 0 . 0009 , * * * * P < 0 . 0001 and ( E ) * P = 0 . 0204 , * * * P = 0 . 0006 , * * * * P < 0 . 0001 . Kruskal - Wallis followed by Dunn ' s multiple comparisons test .

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D, E Quantification of proliferation of 1205Lu (D) and SKMEL5 (E) cells plated for 48 h on plastic, Coll-1 or the indicated fibroblast-derived ECMs prior a 96 h treatment with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi. Cells were counted by Hoechst-labeled nuclei staining. Data are represented as bar plots with mean ± SEM normalized to vehicle of 3 independent experiments. (D) **P=0.015, ***P=0.0009, ****P<0.0001 and (E) *P=0.0204, ***P=0.0006, ****P<0.0001. Kruskal-Wallis followed by Dunn's multiple comparisons test.


F Flow cytometry analysis of cell cycle distribution of SKMEL5 cells cultured on plastic , Coll - 1 or the indicated fibroblast - derived ECMs and treated with vehicle or 2 µM BRAFi combined with 0 . 01 µM MEKi . The percentage of cells in different phases of the cell cycle is indicated .

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F Flow cytometry analysis of cell cycle distribution of SKMEL5 cells cultured on plastic, Coll-1 or the indicated fibroblast-derived ECMs and treated with vehicle or 2 µM BRAFi combined with 0.01 µM MEKi. The percentage of cells in different phases of the cell cycle is indicated.


G Immunoblotting of protein extracts from SKMEL5 cells cultivated as described above on plastic , Coll - 1 or the indicated fibroblast - derived ECMs in the presence or not of BRAFi or BRAFi / MEKi for 96 h , using antibodies against P - ERK1 / 2 , ERK2 or cell cycle markers ( P - Rb , Rb , Cyclin D1 , p27KIP1 , Survivin , p53 ) . HSP60 , loading control .

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G Immunoblotting of protein extracts from SKMEL5 cells cultivated as described above on plastic, Coll-1 or the indicated fibroblast-derived ECMs in the presence or not of BRAFi or BRAFi/MEKi for 96 h, using antibodies against P-ERK1/2, ERK2 or cell cycle markers (P-Rb, Rb, Cyclin D1, p27KIP1, Survivin, p53). HSP60, loading control.


B Immunohistochemical analysis of DDR1 and DDR2 levels on human melanoma tissue microarrays . Representative IHC images and quantification ( right bar histograms ) of DDR1 and DDR2 expression in normal skin , naevus , primary melanoma ( PM ) and lymph node melanoma metastases ( MM ) . Scale bar , 100 µm . Histological scoring of the samples was performed in blinded fashion . Samples were scored as low , medium or high for DDR1 or DDR2 expression ( naevus , n = 12 ; PM , n = 30 ; MM , n = 20 ) .

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B Immunohistochemical analysis of DDR1 and DDR2 levels on human melanoma tissue microarrays. Representative IHC images and quantification (right bar histograms) of DDR1 and DDR2 expression in normal skin, naevus, primary melanoma (PM) and lymph node melanoma metastases (MM). Scale bar, 100 µm. Histological scoring of the samples was performed in blinded fashion. Samples were scored as low, medium or high for DDR1 or DDR2 expression (naevus, n = 12; PM, n = 30; MM, n = 20).


Immunoblotting of equal amounts of protein extracts from melanoma cell lines ( C ) using antibodies against DDR1 , DDR2 or markers of the melanoma cell differentiation AXL , MITF or SOX10 . ERK2 , loading control .

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Immunoblotting of equal amounts of protein extracts from melanoma cell lines (C) using antibodies against DDR1, DDR2 or markers of the melanoma cell differentiation AXL, MITF or SOX10. ERK2, loading control.


Immunoblotting of equal amounts of protein extracts patient - derived short - term cell cultures ( D ) using antibodies against DDR1 , DDR2 or markers of the melanoma cell differentiation AXL , MITF or SOX10 . ERK2 , loading control .

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Immunoblotting of equal amounts of protein extracts patient-derived short-term cell cultures (D) using antibodies against DDR1, DDR2 or markers of the melanoma cell differentiation AXL, MITF or SOX10. ERK2, loading control.


B Immunoblotting of protein extracts from SKMEL5 cells transfected with siCTRL or the combination siDDR1 # 2 / siDDR2 # 2 prior being cultivated on FRC - or MAF - derived ECMs ( left and right panels , respectively ) and treated with vehicle , 5 µM BRAFi or 2 µM BRAFi plus 0 . 01 µM MEKi , using antibodies against the indicated proteins . HSP60 , loading control .

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B Immunoblotting of protein extracts from SKMEL5 cells transfected with siCTRL or the combination siDDR1#2/siDDR2#2 prior being cultivated on FRC- or MAF-derived ECMs (left and right panels, respectively) and treated with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi, using antibodies against the indicated proteins. HSP60, loading control.


D Immunoblotting of protein extracts from 1205Lu , SKMEL5 and MM099 cells plated for 48 h on FRC - derived ECM prior a 96 h treatment with vehicle or 5 µM BRAFi in the presence or not of Imatinib ( 7 µM for 1205Lu , 10 µM for SKMEL5 and MM099 ) or DDR1 - IN - 1 ( 1 µM for 1205Lu , 5 µM for SKMEL5 , 3 µM for MM099 ) , using antibodies against the indicated proteins . HSP60 , loading control .

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D Immunoblotting of protein extracts from 1205Lu, SKMEL5 and MM099 cells plated for 48 h on FRC-derived ECM prior a 96 h treatment with vehicle or 5 µM BRAFi in the presence or not of Imatinib (7 µM for 1205Lu, 10 µM for SKMEL5 and MM099) or DDR1-IN-1 (1 µM for 1205Lu, 5 µM for SKMEL5, 3 µM for MM099), using antibodies against the indicated proteins. HSP60, loading control.


A Representative images of 1205 cells cultivated on collagen - I ( Coll - I ) or FRC - derived ECM for 48 h prior treatment with vehicle or 5 µM BRAFi or 2 µM BRAFi plus 0 . 01 µM MEKi for 96 h . Immunofluorescence for Phospho - DDR1 ( P ( Y792 ) - DDR1 ) ( red ; left panels ) and Phospho - DDR1 / 2 ( P ( Y796 ) - DDR1 / P ( Y740 ) - DDR2 ) ( red ; right panels ) , F - actin ( green ) and nuclei ( blue ) is shown . Enlarged images of P - DDR1 and P - DDR1 / 2 immunostaining are shown . White arrows indicate P - DDR1 and P - DDR1 / 2 cell membrane linear clustering . Scale bar , 25 µm ( enlarged images : 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of 1205 cells cultivated on collagen-I (Coll-I) or FRC-derived ECM for 48 h prior treatment with vehicle or 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi for 96 h. Immunofluorescence for Phospho-DDR1 (P(Y792)-DDR1) (red; left panels) and Phospho-DDR1/2 (P(Y796)-DDR1/P(Y740)-DDR2) (red; right panels), F-actin (green) and nuclei (blue) is shown. Enlarged images of P-DDR1 and P-DDR1/2 immunostaining are shown. White arrows indicate P-DDR1 and P-DDR1/2 cell membrane linear clustering. Scale bar, 25 µm (enlarged images: scale bar, 10 µm).


E Kaplan - Meier survival curves of mice treated with vehicle , Imatinib , BRAFi or BRAFi plus Imatinib . Median time to progression was 18 , 20 , 36 and 48 days , respectively . Log rank ( Mantel - Cox ) for BRAFi vs BRAFi / Imatinib mesylate . * * * * P < 0 . 0001 and hazard ratio ( log rank ) : 0 . 2403 ( 95 % CI of ratio , 0 . 08123 to 0 . 7106 ) .

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

E Kaplan-Meier survival curves of mice treated with vehicle, Imatinib, BRAFi or BRAFi plus Imatinib. Median time to progression was 18, 20, 36 and 48 days, respectively. Log rank (Mantel-Cox) for BRAFi vs BRAFi/Imatinib mesylate. ****P<0.0001 and hazard ratio (log rank): 0.2403 (95% CI of ratio, 0.08123 to 0.7106).


A Sections of 1205Lu xenografts from Fig . 6B were stained with picrosirius red and imaged under transmission light ( upper panels ) or polarized light ( middle panels ) ( scale bar , 500 µm ) or imaged by second harmonic generation ( SHG ) microscopy ( lower panels ) ( scale bar , 50 µm ) to examine collagen fiber network upon the mono or combined regimens .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Sections of 1205Lu xenografts from Fig. 6B were stained with picrosirius red and imaged under transmission light (upper panels) or polarized light (middle panels) (scale bar, 500 µm) or imaged by second harmonic generation (SHG) microscopy (lower panels) (scale bar, 50 µm) to examine collagen fiber network upon the mono or combined regimens.


A Immunoblot analysis of protein extracts from siCTRL - or siDDR1 / 2 - transfected SKMEL5 cells plated on FRC - or MAF - derived ECMs in the presence or not of 5 µM BRAFi , 2 µM BRAFi plus 0 . 01 µM MEKi for 96 h using antibodies against DDR1 , DDR2 , P - ERK1 / 2 , ERK2 , RelB , p100 / p52 and HSP60 as loading control were used .

A Immunoblot analysis of protein extracts from siCTRL- or siDDR1/2-transfected SKMEL5 cells plated on FRC- or MAF-derived ECMs in the presence or not of 5 µM BRAFi, 2 µM BRAFi plus 0.01 µM MEKi for 96 h using antibodies against DDR1, DDR2, P-ERK1/2, ERK2, RelB, p100/p52 and HSP60 as loading control were used.


F , G Immunoblot analysis of protein extracts obtained from SKMEL5 ( F ) or MM099 ( G ) cells plated on FRC - or MAF - derived ECMs and treated with 5 µM BRAFi in combination or not with DDR1 - IN - 1 ( 5 µM for SKMEL5 and 3 µM for MM099 ) or 10 µM NIK inhibitor ( NIKi ) for 96 h . Antibodies against P - ERK1 / 2 , ERK2 , P - Rb , Rb , Survivin , Caspase 3 , cleaved Caspase 3 , RelB , p100 / p52 and HSP60 as loading control were used .

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

F, G Immunoblot analysis of protein extracts obtained from SKMEL5 (F) or MM099 (G) cells plated on FRC- or MAF-derived ECMs and treated with 5 µM BRAFi in combination or not with DDR1-IN-1 (5 µM for SKMEL5 and 3 µM for MM099) or 10 µM NIK inhibitor (NIKi) for 96 h. Antibodies against P-ERK1/2, ERK2, P-Rb, Rb, Survivin, Caspase 3, cleaved Caspase 3, RelB, p100/p52 and HSP60 as loading control were used.


Expression of ERα in control ( shCtrl ) and knockdown ( shERα ) BM67 cells was determined by western blotting ( β - actin served as a loading control ) . Gene expression was determined using polysome - profiling which quantifies both total mRNA and efficiently translated polysome - associated mRNA ( n = 3 ) .

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

Expression of ERα in control (shCtrl) and knockdown (shERα) BM67 cells was determined by western blotting (β-actin served as a loading control). Gene expression was determined using polysome-profiling which quantifies both total mRNA and efficiently translated polysome-associated mRNA (n = 3).


Distributions ( quantified by kernel density estimation ) of p - values for differential expression ( shERα vs . shCtrl BM67 cells ) using data from polysome - associated mRNA ( orange ) , cytosolic mRNA ( purple ) or from analysis of changes in translational efficiency ( i . e . changes in polysome - associated mRNA not paralleled by changes in cytosolic mRNA ) leading to altered protein levels ( red ; a higher density of low p - values indicates a higher frequency of changes in gene expression ) .

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

Distributions (quantified by kernel density estimation) of p-values for differential expression (shERα vs. shCtrl BM67 cells) using data from polysome-associated mRNA (orange), cytosolic mRNA (purple) or from analysis of changes in translational efficiency (i.e. changes in polysome-associated mRNA not paralleled by changes in cytosolic mRNA) leading to altered protein levels (red; a higher density of low p-values indicates a higher frequency of changes in gene expression).


Scatterplot of polysome - associated mRNA vs . cytosolic mRNA log2 fold - changes ( shERα vs . shCtrl ) . Areas of the plot are colored according to the density of data points ( genes ; dark blue corresponds to areas with many

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

Scatterplot of polysome-associated mRNA vs. cytosolic mRNA log2 fold-changes (shERα vs. shCtrl). Areas of the plot are colored according to the density of data points (genes; dark blue corresponds to areas with many genes


Same plot as in ( B ) but including a blue density of p - values from the analysis of translational offsetting .

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

Same plot as in (B) but including a blue density of p-values from the analysis of translational offsetting.


A scatter plot similar to ( C ) but where genes are colored according to their mode of regulation derived from anota2seq analysis . A relaxed threshold ( p < 0 . 05 ) was used to identify a set of transcripts regulated via translation , which did not pass thresholds used for identification of changes in mRNA abundance or translational offsetting ( FDR < 0 . 1 ) . Confidence ellipses ( level 0 . 7 ) are overlaid for each mode of regulation . A bar graph indicates the number of mRNAs regulated via each mode .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 scatter plot similar to (C) but where genes are colored according to their mode of regulation derived from anota2seq analysis. A relaxed threshold (p<0.05) was used to identify a set of transcripts regulated via translation, which did not pass thresholds used for identification of changes in mRNA abundance or translational offsetting (FDR<0.1). Confidence ellipses (level 0.7) are overlaid for each mode of regulation. A bar graph indicates the number of mRNAs regulated via each mode.


Targets from each mode of regulation ( cf . Fig 1E ) were selected for validation by Nanostring . Shown is a scatterplot of Nanostring quantification of polysome - associated mRNA vs . cytosolic mRNA log2 fold - change ( shERα vs . shCtrl ; n = 3 ) . Genes are colored according to their identified mode of regulation ( i . e . from Fig 1E ) . Confidence ellipses ( based on Nanostring data , level 0 . 7 ) are overlaid for each mode of regulation . Selected genes from each mode of regulation are 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Targets from each mode of regulation (cf. Fig 1E) were selected for validation by Nanostring. Shown is a scatterplot of Nanostring quantification of polysome-associated mRNA vs. cytosolic mRNA log2 fold-change (shERα vs. shCtrl; n = 3). Genes are colored according to their identified mode of regulation (i.e. from Fig 1E). Confidence ellipses (based on Nanostring data, level 0.7) are overlaid for each mode of regulation. Selected genes from each mode of regulation are indicated.


Levels of indicated proteins from shERα and control BM67 cells were determined by western blotting with their quantifications . β - actin served as loading control . Graphs represent mean ± standard deviation ( n = 3 - 5 as indicated ) . Protein expression between groups were compared using two - sided paired Student ' s t tests .

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

Levels of indicated proteins from shERα and control BM67 cells were determined by western blotting with their quantifications. β-actin served as loading control. Graphs represent mean ± standard deviation (n = 3-5 as indicated). Protein expression between groups were compared using two-sided paired Student's t tests.


Immunoblotting of BM67 cell lines extract and its quantitation following incubation with 100 µg / mL cycloheximide as indicated . Graphs represent mean ± standard deviation ( n = 5 ) .

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

Immunoblotting of BM67 cell lines extract and its quantitation following incubation with 100 µg/mL cycloheximide as indicated. Graphs represent mean ± standard deviation (n = 5).


nanoCAGE sequencing was applied to determine transcription start sites in shERα BM67 cells . The number of detected transcription start sites ( peaks ) and RefSeq transcripts when sampling increasing number of RNAseq reads are indicated to evaluate the complexity of nanoCAGE RNAseq libraries .

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

nanoCAGE sequencing was applied to determine transcription start sites in shERα BM67 cells. The number of detected transcription start sites (peaks) and RefSeq transcripts when sampling increasing number of RNAseq reads are indicated to evaluate the complexity of nanoCAGE RNAseq libraries.


Boxplots for offset and non - offset mRNAs comparing 5 ' UTR weighted lengths ( log2 scale ) , GC content ( % ) , free energy ( log10 scale , kcal / mole ) . In boxplots , the solid horizontal middle line is the median ; the lower and upper box limits correspond to the first and third quartiles ; the whiskers extend from the box limits to the most extreme value but not further than + / - 1 . 5 x the inter - quartile range ( IQR ) from the box limits . The notches extend to 1 . 58 x the IQR / √ n where n is the number of data points . Data beyond the end of the whiskers are plotted as points . Wilcoxon - Mann - Whitney tests ( two - sided ) were used to assess differences between translationally offset and non - offset mRNAs . Number of transcripts harboring at least one ( dark gray ) or no ( light gray ) uORF in a strong Kozak context were visualized as a bar chart and differences were tested using one - sided Fisher ' s exact tests . A global Bonferroni adjustment was applied on p - values . Analysis was performed on nanoCAGE data from 3 replicates of shERα BM67 cells ; the numbers of transcripts in each group are indicated in the figure .

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

Boxplots for offset and non-offset mRNAs comparing 5' UTR weighted lengths (log2 scale), GC content (%), free energy (log10 scale, kcal/mole). In boxplots, the solid horizontal middle line is the median; the lower and upper box limits correspond to the first and third quartiles; the whiskers extend from the box limits to the most extreme value but not further than +/- 1.5 x the inter-quartile range (IQR) from the box limits. The notches extend to 1.58 x the IQR / √n where n is the number of data points. Data beyond the end of the whiskers are plotted as points. Wilcoxon-Mann-Whitney tests (two-sided) were used to assess differences between translationally offset and non-offset mRNAs. Number of transcripts harboring at least one (dark gray) or no (light gray) uORF in a strong Kozak context were visualized as a bar chart and differences were tested using one-sided Fisher's exact tests. A global Bonferroni adjustment was applied on p-values. Analysis was performed on nanoCAGE data from 3 replicates of shERα BM67 cells; the numbers of transcripts in each group are indicated in the figure.


Volcano plot of ERα dependent miRNA expression in BM67 cells quantified by small RNAseq . Down - and up - regulated miRNAs ( FDR < 0 . 2 and fold - change > 1 . 25 ) are colored in blue and red , respectively .

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

Volcano plot of ERα dependent miRNA expression in BM67 cells quantified by small RNAseq. Down- and up-regulated miRNAs (FDR<0.2 and fold-change > 1.25) are colored in blue and red, respectively.


Validation of miRNA expression using qPCR . Normalized expression ( mean ± standard deviation ; n = 3 ) ( B ) and expression fold change ( shERα vs . shCtrl ) obtained from small RNAseq vs . qPCR ( C ) .

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

Validation of miRNA expression using qPCR. Normalized expression (mean ± standard deviation; n = 3) (B) and expression fold change (shERα vs. shCtrl) obtained from small RNAseq vs. qPCR (C).


Percentage of miRNA among small RNAs in shERα and shCtrl BM67 cells ( mean ± standard deviation ; n = 3 ) .

Percentage of miRNA among small RNAs in shERα and shCtrl BM67 cells (mean ± standard deviation; n = 3).


Assessment of the relationship between presence of miRNA target sites and translational offsetting . ( E ) For visualization , mRNAs were assigned angles describing their mode of regulation such that mRNAs with congruently modulated total and polysome - associated mRNA obtain an angle of ~ 45 degrees , whereas offset mRNAs are assigned an angle of ~ 0 degrees . ( F ) The distributions ( quantified by kernel density estimation ) of such angles were then compared between mRNAs whose levels were induced upon ERα depletion , which were also targets of each suppressed miRNAs separately ( blue lines ) ; and the set of background transcripts ( mRNAs induced upon ERα depletion but not targets of suppressed miRNAs ; black ) . ( G ) For such subsets of miRNA targets ( i . e . from F ) , the relative number of offset and non - offset mRNAs ( as determined by anota2seq analysis ) was also compared to the background ( same as in F ) using Fisher ' s exact test ; and the resulting adjusted p - values ( FDRs ; across all assessed miRNAs ) were visualized as a histogram . ( H - I ) A similar assessment as in ( F - G ) but for suppressed mRNAs and induced miRNAs . ( J ) A similar assessment as in ( H ) but for randomly selected groups of 10 miRNAs ( gray lines ) compared to background ( black ) and targets of all induced miRNA ( red ; i . e . from H ) .

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

Assessment of the relationship between presence of miRNA target sites and translational offsetting. (E) For visualization, mRNAs were assigned angles describing their mode of regulation such that mRNAs with congruently modulated total and polysome-associated mRNA obtain an angle of ~45 degrees, whereas offset mRNAs are assigned an angle of ~0 degrees. (F) The distributions (quantified by kernel density estimation) of such angles were then compared between mRNAs whose levels were induced upon ERα depletion, which were also targets of each suppressed miRNAs separately (blue lines); and the set of background transcripts (mRNAs induced upon ERα depletion but not targets of suppressed miRNAs; black). (G) For such subsets of miRNA targets (i.e. from F), the relative number of offset and non-offset mRNAs (as determined by anota2seq analysis) was also compared to the background (same as in F) using Fisher's exact test; and the resulting adjusted p-values (FDRs; across all assessed miRNAs) were visualized as a histogram. (H-I) A similar assessment as in (F-G) but for suppressed mRNAs and induced miRNAs. (J) A similar assessment as in (H) but for randomly selected groups of 10 miRNAs (gray lines) compared to background (black) and targets of all induced miRNA (red; i.e. from H).


For each codon , the average frequency ( per thousand ) is compared between transcripts induced but offset vs non - offset ( i . e . abundance mode of regulation ) upon ERα depletion in BM67 cells . Codons for the same amino acid are connected by a gray line . Heatmap of standardized residuals from a chi - squared contingency table test . Shown in red and blue are cells with counts significantly higher and lower , respectively , than expected counts under the null hypothesis ( i . e . independence between mode for regulation of gene expression and codon composition ) . A correspondence analysis for average codon frequency among transcripts annotated to each GO term . Each gray dot corresponds to one GO term . The gene expression modes observed upon ERα depletion ( Fig 1E ) were then projected into the same dimensions based on the average codon frequencies of included transcripts . A correspondence analysis for codon frequency in each regulated mRNA ( from Fig 1E ) . Each dot represents one mRNA and is colored according to its mode of regulation . Codons identified as over - represented among translationally offset mRNAs are also indicated . Unsupervised clustering of gene level codon usage normalized by amino acid counts . All regulated mRNAs ( from Fig 1E ) are shown in rows and all codons in columns . Codons identified as over - represented among mRNAs whose levels were induced but offset or suppressed but offset are indicated in light and dark blue , respectively .

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

For each codon, the average frequency (per thousand) is compared between transcripts induced but offset vs non-offset (i.e. abundance mode of regulation) upon ERα depletion in BM67 cells. Codons for the same amino acid are connected by a gray line. Heatmap of standardized residuals from a chi-squared contingency table test. Shown in red and blue are cells with counts significantly higher and lower, respectively, than expected counts under the null hypothesis (i.e. independence between mode for regulation of gene expression and codon composition). A correspondence analysis for average codon frequency among transcripts annotated to each GO term. Each gray dot corresponds to one GO term. The gene expression modes observed upon ERα depletion (Fig 1E) were then projected into the same dimensions based on the average codon frequencies of included transcripts. A correspondence analysis for codon frequency in each regulated mRNA (from Fig 1E). Each dot represents one mRNA and is colored according to its mode of regulation. Codons identified as over-represented among translationally offset mRNAs are also indicated. Unsupervised clustering of gene level codon usage normalized by amino acid counts. All regulated mRNAs (from Fig 1E) are shown in rows and all codons in columns. Codons identified as over-represented among mRNAs whose levels were induced but offset or suppressed but offset are indicated in light and dark blue, respectively.


Immunoblotting of BM67 cell extract for ELP3 after stable shRNA - mediated ERα knockdown in BM67 cells . Graphs represent mean ± standard deviation of n = 3 .

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

Immunoblotting of BM67 cell extract for ELP3 after stable shRNA-mediated ERα knockdown in BM67 cells. Graphs represent mean ± standard deviation of n=3.


qPCR analysis and immunoblotting of BM67 cell extract for indicated proteins with its quantitation following deletion of Esr1 gene by CRISPR - Cas9 technology . Graphs represent mean ± standard deviation of n = 5 .

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

qPCR analysis and immunoblotting of BM67 cell extract for indicated proteins with its quantitation following deletion of Esr1 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=5.


qPCR analysis and immunoblotting of BM67 cell extract for ELP3 and DEK with its quantitation following deletion of Elp3 gene by CRISPR - Cas9 technology . Graphs represent mean ± standard deviation of n = 4 .

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

qPCR analysis and immunoblotting of BM67 cell extract for ELP3 and DEK with its quantitation following deletion of Elp3 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=4.


Immunoblotting and qPCR analysis comparing cell extracts obtained from BM67 cells transduced with pLentiCRISPRV2 empty vector , Elp3 - null BM67 cells and Elp3 - null BM67 cells overexpressing ELP3 protein containing silent mutations in the sgRNA binding site . Graphs represent mean ± standard deviation of n = 3 - 4 .

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

Immunoblotting and qPCR analysis comparing cell extracts obtained from BM67 cells transduced with pLentiCRISPRV2 empty vector, Elp3-null BM67 cells and Elp3-null BM67 cells overexpressing ELP3 protein containing silent mutations in the sgRNA binding site. Graphs represent mean ± standard deviation of n=3-4.


Boxplots ( plotted as in Fig 3B ) of gene expression of ELP3 , ALKBH8 and CTU2 upon treatment with 17β - E2 and / or ICI - 182780 in MCF7 cells ( extracted from ( Wardell et al , 2012 ; Data Ref : McDonnell et al , 2012 ) ) . Wilcoxon - Mann - Whitney tests ( two - sided ) were used to assess differences between conditions ( n = 10 ) and a global Bonferroni adjustment was applied on p - values .

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

Boxplots (plotted as in Fig 3B) of gene expression of ELP3, ALKBH8 and CTU2 upon treatment with 17β-E2 and/or ICI-182780 in MCF7 cells (extracted from (Wardell et al, 2012; Data Ref: McDonnell et al, 2012)). Wilcoxon-Mann-Whitney tests (two-sided) were used to assess differences between conditions (n = 10) and a global Bonferroni adjustment was applied on p-values.


Immunoblotting of MCF7 cell extracts . MCF - 7 cells were grown in phenol red - free RPMI media supplemented with 5 % charcoal - stripped serum for 24 hours and then treated with 1 nM E2 and / or 100 nM ICI - 182780 for 24 hours . Graphs represent mean ± SD of n = 5 experiments . Data were analyzed using two - sided paired Student ' s t tests .

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

Immunoblotting of MCF7 cell extracts. MCF-7 cells were grown in phenol red-free RPMI media supplemented with 5% charcoal-stripped serum for 24 hours and then treated with 1 nM E2 and/or 100 nM ICI-182780 for 24 hours. Graphs represent mean ± SD of n=5 experiments. Data were analyzed using two-sided paired Student's t tests.


ChIP - seq reveals ER binding sites proximal to the ELP3 gene . Each track represents a distinct ChIP - seq dataset .

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

ChIP-seq reveals ER binding sites proximal to the ELP3 gene. Each track represents a distinct ChIP-seq dataset.


Immunoblotting of MCF - 7 cell extract for indicated proteins following deletion of ELP3 gene by CRISPR - Cas9 technology .

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

Immunoblotting of MCF-7 cell extract for indicated proteins following deletion of ELP3 gene by CRISPR-Cas9 technology.


Quantification of mcm5S2 - U , mcm5U and cm5U levels in ELP3 - null MCF - 7 cells and in MCF - 7 cells treated with 100 nM ICI - 182780 for 72 hours versus their respective controls by liquid chromatography - mass spectrometry ( LC - MS ) analysis . The MCF - 7 cells were grown in phenol red - free RPMI media supplemented with 5 % charcoal - stripped serum for 24 hours prior to ICI - 182780 treatment . Graphs represent mean ± SD of n = 4 . Data were analyzed using two - sided paired Student ' s t tests . Vh , Vehicle ; ICI , ICI - 182780 .

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

Quantification of mcm5S2-U, mcm5U and cm5U levels in ELP3-null MCF-7 cells and in MCF-7 cells treated with 100 nM ICI-182780 for 72 hours versus their respective controls by liquid chromatography-mass spectrometry (LC-MS) analysis. The MCF-7 cells were grown in phenol red-free RPMI media supplemented with 5% charcoal-stripped serum for 24 hours prior to ICI-182780 treatment. Graphs represent mean ± SD of n=4. Data were analyzed using two-sided paired Student's t tests. Vh, Vehicle; ICI, ICI-182780.


( a ) Nuclear localization of HLH - 30 was visualized by fluorescence microscopy in day 1 adult wild - type ( WT ) ( upper panel ) and glp - 1 ( e2141 ) ( lower panel ) animals expressing HLH - 30 : : GFP raised at the non - permissive temperature ( 25 ° C ) . Inserts show enlarged intestinal cells . Graph shows percentage of animals with HLH - 30 in the nuclei of intestinal cells ( four biological replicates , ~ 50 animals each , mean ± s . d . , * * P0 . 01 , Student ' s t - test ) . Magnification × 100 ; scale bar , 100 μm .

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(a) Nuclear localization of HLH-30 was visualized by fluorescence microscopy in day 1 adult wild-type (WT) (upper panel) and glp-1(e2141) (lower panel) animals expressing HLH-30::GFP raised at the non-permissive temperature (25°C). Inserts show enlarged intestinal cells. Graph shows percentage of animals with HLH-30 in the nuclei of intestinal cells (four biological replicates, ~50 animals each, mean±s.d., **P0.01, Student's t-test). Magnification × 100; scale bar, 100 μm.


( b ) Expression of hlh - 30 and putative autophagy - related and lysosomal target genes was measured by quantitative PCR ( qPCR ) in day 1 adult WT and glp - 1 ( e2141 ) animals raised at 25 ° C ( mean ± s . d . of three biological replicates , * P0 . 05 , * * P0 . 01 , Student ' s t - test ) .

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(b) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by quantitative PCR (qPCR) in day 1 adult WT and glp-1(e2141) animals raised at 25°C (mean±s.d. of three biological replicates, *P0.05, **P0.01, Student's t-test).


( c ) Expression of hlh - 30 and putative autophagy - related and lysosomal target genes was measured by qPCR in day 1 adult glp - 1 ( e2141 ) and glp - 1 ( e2141 ) ; hlh - 30 ( tm1978 ) animals raised at 25 ° C ( mean ± s . d . of three biological replicates , * P0 . 05 , * * P0 . 01 , Student ' s t - test ) . ctsa * is a cathepsin A orthologue ( cosmid C08H9 . 1 ( ref . 30 ) ) . See Supplementary Fig . 1 for qPCR analyses of hlh - 30 ( tm1978 ) ( control for c ) and WT and glp - 1 ( e2141 ) animals fed bacteria expressing hlh - 30 dsRNA .

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(c) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by qPCR in day 1 adult glp-1(e2141) and glp-1(e2141); hlh-30(tm1978) animals raised at 25°C (mean±s.d. of three biological replicates, *P0.05, **P0.01, Student's t-test). ctsa* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)). See Supplementary Fig. 1 for qPCR analyses of hlh-30(tm1978) (control for c) and WT and glp-1(e2141) animals fed bacteria expressing hlh-30 dsRNA.


( d , e ) GFP : : LGG - 1 punctae were quantified in ( d ) hypodermal seam cells or ( e ) proximal intestinal cells of L3 larvae of WT and glp - 1 ( e2141 ) animals . Animals were fed bacteria expressing control or hlh - 30 dsRNA for two generations at 20 ° C . Eggs were then transferred to plates seeded with the appropriate dsRNA - expressing bacteria at 25 ° C and analysed at the L3 larval stage ( mean ± s . e . m . of ~ 300 seam cells and ~ 25 intestines , * * P0 . 01 , Student ' s t - test ) .

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(d,e) GFP::LGG-1 punctae were quantified in (d) hypodermal seam cells or (e) proximal intestinal cells of L3 larvae of WT and glp-1(e2141) animals. Animals were fed bacteria expressing control or hlh-30 dsRNA for two generations at 20 °C. Eggs were then transferred to plates seeded with the appropriate dsRNA-expressing bacteria at 25 °C and analysed at the L3 larval stage (mean±s.e.m. of ~300 seam cells and ~25 intestines, **P0.01, Student's t-test).


( f , g ) glp - 1 ( e2141 ) animals expressing ( f ) LMP - 1 : : GFP or ( g ) SQST - 1 : : GFP were raised at the non - permissive temperature ( 25 ° C ) and fed bacteria expressing control or hlh - 30 dsRNA from hatching . Micrographs of the posterior intestine were taken on day 1 of adulthood , and LMP - 1 : : GFP fluorescence ( mean ± s . d . of ~ 10 animals , * * P0 . 01 , Student ' s t - test ) and SQST - 1 : : GFP foci ( mean ± s . d . of ~ 30 animals , * * P0 . 01 , Student ' s t - test ) were quantified . Experiments were performed at least three times with similar results . See Supplementary Fig . S2a , c for images of whole animals and Supplementary Fig . S2b , d for replicates . Magnification , × 200 ; scale bar , 100 μm .

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(f,g) glp-1(e2141) animals expressing (f) LMP-1::GFP or (g) SQST-1::GFP were raised at the non-permissive temperature (25°C) and fed bacteria expressing control or hlh-30 dsRNA from hatching. Micrographs of the posterior intestine were taken on day 1 of adulthood, and LMP-1::GFP fluorescence (mean±s.d. of ~10 animals, **P0.01, Student's t-test) and SQST-1::GFP foci (mean±s.d. of ~30 animals, **P0.01, Student's t-test) were quantified. Experiments were performed at least three times with similar results. See Supplementary Fig. S2a,c for images of whole animals and Supplementary Fig. S2b,d for replicates. Magnification, × 200; scale bar, 100 μm.


( a ) Nuclear localization of HLH - 30 was quantified in day 1 adult animals expressing HLH - 30 : : GFP . Animals were fed bacteria expressing control or tor dsRNA from hatching and raised at 20 ° C ( three biological replicates , ~ 50 animals each , mean ± s . d . , * * P0 . 01 , Student ' s t - test ) .

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(a) Nuclear localization of HLH-30 was quantified in day 1 adult animals expressing HLH-30::GFP. Animals were fed bacteria expressing control or tor dsRNA from hatching and raised at 20 °C (three biological replicates, ~50 animals each, mean±s.d., **P0.01, Student's t-test).


b , c ) Expression of putative autophagy - related and lysosomal target genes was measured by quantitative PCR in day 1 ( b ) WT ( N2 ) and ( c ) hlh - 30 ( tm1978 ) animals fed bacteria expressing control or tor dsRNA from hatching ( 20 ° C ) . Data are mean ± s . d . of biological triplicates . * P0 . 05 , * * P0 . 01 ; Student ' s t - test . csta * is a cathepsin A orthologue ( cosmid C08H9 . 1 ( ref . 30 ) ) .

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b,c) Expression of putative autophagy-related and lysosomal target genes was measured by quantitative PCR in day 1 (b) WT (N2) and (c) hlh-30(tm1978) animals fed bacteria expressing control or tor dsRNA from hatching (20 °C). Data are mean±s.d. of biological triplicates. *P0.05, **P0.01; Student's t-test. csta* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)).


( d ) Lifespan analysis of WT animals and hlh - 30 ( tm1978 ) mutants fed bacteria expressing control or tor dsRNA from day 1 of adulthood was carried out at 20 ° C . See Supplementary Table S3 for details of lifespan analyses and replicate experiments .

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(d) Lifespan analysis of WT animals and hlh-30(tm1978) mutants fed bacteria expressing control or tor dsRNA from day 1 of adulthood was carried out at 20 °C. See Supplementary Table S3 for details of lifespan analyses and replicate experiments.


Lifespan analyses of ( a ) WT ( N2 ) and ( b ) germline - less glp - 1 ( e2141 ) animals raised at the non - permissive temperature ( 25 ° C ) and fed bacteria expressing control or hlh - 30 dsRNA from day 1 of adulthood were carried out at 20 ° C . Lifespan analyses of ( c ) dietary - restricted eat - 2 ( ad1116 ) mutants , ( d ) insulin / IGF - 1 receptor daf - 2 ( e1370 ) mutants , ( e ) mitochondrial respiration clk - 1 ( e2519 ) mutants and ( f ) mRNA translation rsks - 1 ( sv31 ) mutants , fed bacteria expressing control or hlh - 30 dsRNA from day 1 of adulthood ( c , d , f ) or larval L4 stage ( e ) , were carried out at 20 ° C . See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments .

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Lifespan analyses of (a) WT (N2) and (b) germline-less glp-1(e2141) animals raised at the non-permissive temperature (25 °C) and fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood were carried out at 20 °C. Lifespan analyses of (c) dietary-restricted eat-2(ad1116) mutants, (d) insulin/IGF-1 receptor daf-2(e1370) mutants, (e) mitochondrial respiration clk-1(e2519) mutants and (f) mRNA translation rsks-1(sv31) mutants, fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood (c,d,f) or larval L4 stage (e), were carried out at 20 °C. See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments.


( a ) Nuclear localization of HLH - 30 was quantified in day 1 adult WT , eat - 2 , daf - 2 , clk - 1 and rsks - 1 mutants ( mean ± s . d . of four biological replicates , ~ 50 animals each , * * P0 . 01 , analysis of variance ) .

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(a) Nuclear localization of HLH-30 was quantified in day 1 adult WT, eat-2, daf-2, clk-1 and rsks-1 mutants (mean±s.d. of four biological replicates, ~50 animals each, **P0.01, analysis of variance).


( b ) Expression of hlh - 30 was measured by quantitative PCR ( qPCR ) in day 1 adult WT ( N2 ) , eat - 2 ( ad1116 ) ( dietary restriction , DR ) , daf - 2 ( e1370 ) ( insulin / IGF - 1 signaling ) , clk - 1 ( e2519 ) ( mitochondrial respiration ) and rsks - 1 ( sv31 ) ( mRNA translation ) animals ( mean ± s . d . of three biological replicates , * P0 . 05 , Student ' s t - test ) .

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(b) Expression of hlh-30 was measured by quantitative PCR (qPCR) in day 1 adult WT (N2), eat-2(ad1116) (dietary restriction, DR), daf-2(e1370) (insulin/IGF-1 signaling), clk-1(e2519) (mitochondrial respiration) and rsks-1(sv31) (mRNA translation) animals (mean±s.d. of three biological replicates, *P0.05, Student's t-test).


( c ) Lifespan analysis of WT and transgenic animals overexpressing HLH - 30 : : GFP , and raised and maintained on OP50 was carried out at 20 ° C . See Supplementary Table S7 for details of lifespan analyses and replicate experiments .

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(c) Lifespan analysis of WT and transgenic animals overexpressing HLH-30::GFP, and raised and maintained on OP50 was carried out at 20 °C. See Supplementary Table S7 for details of lifespan analyses and replicate experiments.


( d ) GFP : : LGG - 1 punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH - 30 ( n > 300 , ± s . e . m . , * * P0 . 01 , Student ' s t - test ) . The increase in GFP : : LGG - 1 punctae observed in animals overexpressing HLH - 30 could be reversed by atg - 18 RNAi treatment ( data not shown ) .

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(d) GFP::LGG-1 punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH-30 (n>300, ±s.e.m., **P0.01, Student's t-test). The increase in GFP::LGG-1 punctae observed in animals overexpressing HLH-30 could be reversed by atg-18 RNAi treatment (data not shown).


( e ) TFEB expression was measured by qPCR in the livers of 4 . 5 - month - old female ( F ) and male ( M ) mice fed AL or subjected to DR for 5 . 5 weeks starting at 3 months of age ( mean ± s . e . m . of ~ 20 mice per group , * P0 . 05 , Student ' s t - test ) .

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(e) TFEB expression was measured by qPCR in the livers of 4.5-month-old female (F) and male (M) mice fed AL or subjected to DR for 5.5 weeks starting at 3 months of age (mean±s.e.m. of ~20 mice per group, *P0.05, Student's t-test).


( f ) TFEB protein was detected by western blotting of nuclear fractions from the livers of five female and five male mice fed AL or subjected to DR . Actin was included as a loading control , see Supplementary Fig . S8 for additional controls . ( g ) TFEB protein levels in Fig . 4f were quantified by densitometry and normalized to actin ( mean ± s . d . , * P0 . 05 , Student ' s t - test ) .

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(f) TFEB protein was detected by western blotting of nuclear fractions from the livers of five female and five male mice fed AL or subjected to DR. Actin was included as a loading control, see Supplementary Fig. S8 for additional controls. (g) TFEB protein levels in Fig. 4f were quantified by densitometry and normalized to actin (mean±s.d., *P0.05, Student's t-test).


B ) SUMO2 immunoprecipitation using crosslinked SUMO2 antibody beads and liver samples of wild - type C57BL6 / J mice in the fasted ( 16 h ) or re - fed ( 16 h fasted and re - fed 2 h ) states . Eluates analyzed by immunoblotting using anti - Prox1 antibodies and ponceau staining was used as loading control . Data information Data : mean ± SEM . Significance was determined by one - way ANOVA with Dunnett ´ s multiple comparison test relative to samples collected at ZT 12 . * * * P ≤ 0 . 001 .

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B) SUMO2 immunoprecipitation using crosslinked SUMO2 antibody beads and liver samples of wild-type C57BL6/J mice in the fasted (16 h) or re-fed (16 h fasted and re-fed 2 h) states. Eluates analyzed by immunoblotting using anti-Prox1 antibodies and ponceau staining was used as loading control. Data information Data: mean ±SEM. Significance was determined by one-way ANOVA with Dunnett´s multiple comparison test relative to samples collected at ZT 12. *** P≤0.001.


C ) 8 weeks old C57BL / 6N male mice were fasted ( food removed at ZT 12 ) or re - fed ( fasted from ZT4 to ZT12 for synchronization ; food re - introduced at ZT 12 ) . Tissue samples were collected at ZT 12 , 15 , 18 and 21 ( n = 4 ) . Liver lysates analyzed by immunoblotting using anti - Prox1 and anti - P _ S6K ( Thr389 ) antibodies ; β actin was detected as an input control . The quantification of SUMOylated Prox1 ( % ) and total Prox1 protein expression as well as Prox1 mRNA levels analyzed by qPCR are shown . qPCR data is presented as relative fold change normalized to the housekeeping gene TBP . Data information : Every dot represents one individual mouse . Data : mean ± SEM . Significance was determined by one - way ANOVA with Dunnett ´ s multiple comparison test relative to samples collected at ZT 12 . * * * P ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 8 weeks old C57BL/6N male mice were fasted (food removed at ZT 12) or re-fed (fasted from ZT4 to ZT12 for synchronization; food re-introduced at ZT 12). Tissue samples were collected at ZT 12, 15, 18 and 21 (n=4). Liver lysates analyzed by immunoblotting using anti-Prox1 and anti-P_S6K(Thr389) antibodies; β actin was detected as an input control. The quantification of SUMOylated Prox1 (%) and total Prox1 protein expression as well as Prox1 mRNA levels analyzed by qPCR are shown. qPCR data is presented as relative fold change normalized to the housekeeping gene TBP. Data information: Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by one-way ANOVA with Dunnett´s multiple comparison test relative to samples collected at ZT 12. *** P≤0.001.


B ) HepG2 cells overexpressing HA - tagged mouse wild - type Prox1 ( wt ) , Prox1 K556R mutant or Prox1 E558A mutant . Cell lysates analyzed by immunoblotting using anti - HA antibodies ; tubulin was detected as an input control .

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B) HepG2 cells overexpressing HA-tagged mouse wild-type Prox1 (wt), Prox1 K556R mutant or Prox1 E558A mutant. Cell lysates analyzed by immunoblotting using anti-HA antibodies; tubulin was detected as an input control.