EMBO/SourceData huggingface dataset:
Figure 4A 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.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).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).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).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).
Figure 5A 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).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).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).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).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).
Figure 6A 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.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).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).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.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).
Figure 7A 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).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).
Figure 1(A) Western blot analysis of NDUFS3 protein levels in homogenates from quadriceps muscle of 15-days old (upper panel) and 1-month old animals (lower panel).(B) Body weight comparison over time of Ndufs3 smKO male mice (filled blue squares; n=14) and age matched wild-type (WTFlx; n=15) males (empty blue squares). Ndufs3 smKO females (filled red circles) showed similar results (n=10 for smKO, and n=12 for WTFlx group). P values were calculated by Student's t test;(C) Representative image of a smKO male Ndfu3s3-smKO mouse and a WTFlx littermate of 6-month old showing decreased body weight.(D) Survival curve of Ndufs3 smKO female mice (red line) and males (blue line). P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0010 for males (n=19), and p=0.0004 for females (n=10).(E) Nocturnal ambulatory activity of Ndufs3 smKO and aged matched wild-type littermates from 1 and 2 months old male mice. P values were calculated by Student's t test. P values were calculated by Student's t test to determine the level of statistical difference. ; *p<0.05, **p<0.01 and ***p<0.0001.(F) The number of falls in the treadmill of WTFlx and Ndufs3 smKO male mice, starting at 2 months (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001.(G) Rotarod performed by Ndufs3 smKO and wild-type male mice. (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001.
Figure 2(A) Representative image of quadriceps from WTFlx and Ndufs3 smKO animals of 8 months of age. Muscle weight from Ndufs3 smKO males (black bar) and wild-type mice (white bar) from 6, and 8 months of age (n=5 each group). Bars represent means ± standard error (SEM).(B) Representative H&E, COX, and SDH staining of quadriceps from WTFlx and Ndufs3-Mlc1f smKO at 8 months old.(C) Cross-sectional area (CSA) of quadriceps muscle fibers. A minimum of 100 fibers/sample were analyzed (n =3/genotype). The percentage of central nuclei in skeletal muscle fibers of quadriceps muscle of WTFlx and Ndufs3 smKO at 8 months (n =3 mice for WTFlx and n=4 mice for Ndufs3 smKO). P values were calculated by Student's t test.(D) Serum lactate levels of WTFlx and Ndufs3-smKO animals at different ages. Ndufs3 smKO male mice (filled black squares) showed increased levels compared to matched WTFlx males (empty black squares). Error bars represent SEM.(E) BN-PAGE In-Gel Activities of Complex I and IV measured in homogenates from quadriceps of wild-type and Ndufs3-Mlc1f smKO animals at 2 and 4 months.(F-G) Western blots of muscle homogenates (quadriceps) of WTFlx and Ndufs3-Mlc1f smKO animals at different ages (2 and 4 months, respectively) using antibodies against PGC-1α, COX1 (complex IV subunit), NDUFS3, VDAC1, SDHA (complex II subunit), and β-actin. (H) Quantification of the western blots in panels F and G. Bars represent means ±SEM of n=4 for each group. P values were determined by Student's t test.
Figure 3(C) The KO-GFP mouse was visibly smaller and weaker than a KO-NDUFS3 mouse, at 7 months.(D) Females body weight injected with rAAV9-Ndufs3 (heretofore referred to as KO-NDUFS3) mice were similar to WTFlx mice body weight and significantly higher than the rAAV9-eGFP injected mice (KO-GFP) starting at 5 months. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.(E) Males body weight of KO-NDUFS3 mice were similar to WTFlx mice, and significantly different from KO-GFP from 4 months onwards. Statistical analysis was performed by One-way ANOVA followed by a Tukey post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.(F) Treadmill performance was significantly improved in the KO-NDUFS3 mice when compared to the KO-GFP, from 2 months onwards. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.(G) Four limb strength was evaluated by the grid hang test. KO-NDUFS3 mice performed significantly better starting at 3 months, when compared to the KO-GFP mice. F) and G) Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.(H) Quadriceps and gastrocnemius wet weight of 6 months old mice was similar between WTFlx and KO-NDUFS3 mice. Quadriceps weight was significantly increased in KO-NDUFS3 mice when compared to KO-GFP injected. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. ns- not significant. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.(I) Survival curve of Ndufs3 smKO mice injected with rAVV9-Ndufs3 (blue line) and with rAVV9-eGFP (green line). WTFlx mice are depicted in black. rAAV9-Ndufs3 injections extended the survival of the smKO mice.
Figure 4(A) Western blots of 6 months old quadriceps of WTFlx, KO and KO-NDUFS3 injected mice at 15-18 days old. GFP expression was observed only in the KO-GFP mice. Protein loading was obtained with stain-free gel technology. (B) Quantification of NDUFS3, COXI and SDHA expression levels in female mice showed in A.(C) Western blot of BN-PAGE of quadriceps of 6-month-old mice. We analyzed: CI (NDUFA9, NDUFB8), CIV (COX1), CII (SDHA). (D) Quantification of the BN-PAGE western blots from female mice shown in panel C.(E) BN-PAGE in-gel activity of CI and CIV in quadriceps of 6-month-old mice. (F) Quantification of BN-PAGE in-gel activities in females.(G) Total mtDNA levels in quadriceps from males and females were determined by qPCR using the ratio ND1/18S. Inset shows the appearance of the quadriceps, reflecting normalization of mitochondrial levels after replacement of NDUFS3.(H) Spectrophotometric CI/citrate synthase activity ratio was measured in quadriceps homogenates of 6-month-old males and females.(I) Plasma lactate levels were determined as described in methods. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test.
Figure 5(B) The smKO mice group that received retro-orbital injections at 2 months was tested prior to the rAAV9-Ndufs3 injection showing a significant exercise intolerance attested by the number of falls in the treadmill. The KO-NDUFS3 gradually recovered over time. Individual KO-NDUFS3 mice are color coded. Green triangles represent the animals injected with GFP whereas white triangles represent non-injected mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.(C) The hang test was performed in symptomatic mice which had a significant decrease in four-limb muscle strength and coordination, at 2 months. The KO-NDUFS3 mice showed a significant recovery 3.5 months after injection. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.(D) KO-NDUFS3 muscle weight was similar to WTFlx mice, and it was significantly increased in gastrocnemius when compared to KO-GFP mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.(E) Immunohistochemical analysis with NDUFB8 and COX1 antibodies in quadriceps muscle sections of 6-month-old animals. The expression of NDUFB8 (representative of assembled CI) was recovered in the KO-NDUFS3 mice and COX1 expression was similar to WTFlx animals.(F) COX and COX/SDH activity staining showed that KO-NDUFS3 quadriceps were reverted to levels found in wild-type, at 6 months. n-indicates the number of animals used per group, scale: 100 µm.
Figure 6(A) Western blots showed NDUFS3 expression in KO-NDUFS3 and GFP expression in the KO-GFP quadriceps samples. PGC-1α expression was normalized in the KO-NDUFS3 mice which was abnormally elevated in the KO-GFP. The same was observed for CIV (COX1), CIII (Core 2) and CII (SDHA). (B) Quantification of western blots showed in A. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.(C) BN-PAGE western blots showed recovery of Complex I assembly into complex and supercomplexes in KO-NDUFS3 injected mice. CIV and CII were similar to wild-type in the KO-NDUFS3 samples, at 6 months.(D) Quantification of BN-PAGE western blots Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.(E) BN-PAGE In-gel CI and IV activities of KO-NDUFS3 mice were also reverted to levels similar to the WTFlx samples (F) Quantification of in-gel activities. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.(G) Complex I/Citrate synthase activity of KO-NDUFS3 samples measured spectrophotometrically were similar to WTFlx samples and increased in comparison to KO-GFP mice. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.(H) Total mtDNA levels were determined by qPCR. MtDNA levels in KO-NDUFS3 mice were decreased when compared to KO-GFP mice and not different from the levels in WTFlx mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.(I) The serum lactate levels were similar to WTFlx in KO-NDUFS3 samples and significantly decreased when compared to KO-GFP levels. The individual KO-NDUFS3 animals are color coded. Green triangles represent the animals injected with rAAV9-eGFP whereas black triangles represent animals which did not receive an rAAV9-eGFP injection. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.
Figure 1(B) DNA knotting probability (Pkn) of intracellular chromatin (observed) does not scale proportionally to the length of DNA (expected). The slope of Pkn is reduced in chromatin stretches larger than 20 nucleosomes. Data from (Valdes et al., 2018).
Figure 2(B) Lanes 1 and 2: negatively supercoiled plasmid (YEp24) and its equilibrium distribution of Lk topoisomers upon its relaxation with Topo I. Lanes 3 and 4: distribution of Lk topoisomers of YEp24 upon its relaxation in lysates of ∆top1 TOP2 yeast cells in absence and after the addition of ICRF-193. Plots compare the relative intensity of individual topoisomers of the Lk distributions in lanes 2, 3 and 4.(C) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome present in the lysates of ∆top1 TOP2 yeast cells before and after the addition of ICRF-193 (see details in Fig EV3). Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots (see details in Fig EV4). The graph shows Pkn of YEp13 (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.(E) Lk distributions of the control plasmid (YEp24) relaxed by lysates of ∆top1 top2-4 yeast cells that expressed TOP2 or Top2-∆83. Plots compare the relative intensity of individual Lk topoisomers.(F) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome produced in the presence of TOP2 or Top2-∆83. Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots. Graph: Pkn of YEp13 (mean ± SD from three independent experiments).
Figure 3(A) Top, DNA content (n/2n) of exponentially growing (OD600 = 0.6-0.8) smc2-8 yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). Graph: Pkn of YEp13 before and after the inactivation of condensin.Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (B) Experiments conducted as in (A) but in scc1-73 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of cohesin.Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (C) Experiments conducted as in (A) but in smc6-9 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of the Smc5/6 complex.(D, E) Experiments conducted as in (A), but in cells arrested in G1 with alfa-factor (D) or in metaphase with nocodazole (E) for 2 h at 26oC and for one additional hour at 26oC or 35oC.(F, G) Experiments conducted as in (B), but in cells arrested in G1 with alfa-factor for 2 h at 26oC (F) or in metaphase with nocodazole (G) and for one additional hour at 26oC or 35oC.(H, I) Experiments conducted as in (C), but in cells arrested in G1 with alfa-factor (H) or in metaphase with nocodazole (I) for 2 h at 26oC and for one additional hour at 26oC or 35oC.