Journal: Journal of Healthcare Engineering

Article Title: TSG Targeting KDM5A Affects Osteogenic Differentiation of Bone Mesenchymal Stem Cells Induced by Bone Morphogenetic Protein 2

doi: 10.1155/2022/6472864

Figure Lengend Snippet: KDM5A expression was downregulated under osteogenic differentiation induced by bone morphogenetic protein 2 (BMP-2). (a) The expression level of KDM5A was detected by qPCR. (b) Western blot detection of KDM5A expression. Data were mean ± (S) D, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Small interfering RNA (siRNA) targeting KDM5A (si-KMD5A) and siRNA negative control (si-NC) were all obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot

Journal: Journal of Healthcare Engineering

Article Title: TSG Targeting KDM5A Affects Osteogenic Differentiation of Bone Mesenchymal Stem Cells Induced by Bone Morphogenetic Protein 2

doi: 10.1155/2022/6472864

Figure Lengend Snippet: Knockout of KDM5A promoted osteogenic differentiation of bone marrow mesenchymal stem cells. (a) qRT-PCR analysis of KDM5A expression in transfected MSCs. (b) Alkaline phosphatase staining and activity of MSCs with si-KDM5A were detected by ALP staining 7 days after osteogenesis induction. (c) Alizarin Red S staining was used to detect mineralized nodules of bone marrow mesenchymal stem cells after si-KDM5A. (d) qPCR was used to detect OPN expression in MSCs of si-KDM5A after osteogenesis induction. (e) qPCR detection of OCN expression in MSCs of si-KDM5A after osteogenesis induction. (f) qPCR was used to detect Runx2 expression in MSCs of si-KDM5A after osteogenesis induction. (g) qPCR was used to detect Osterix expression in MSCs of si-KDM5A after osteogenesis induction. (h) qPCR was used to detect Col1a1 expression in MSCs of si-KDM5A after osteogenesis induction. Data were mean ± (S) D, ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Small interfering RNA (siRNA) targeting KDM5A (si-KMD5A) and siRNA negative control (si-NC) were all obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Knock-Out, Quantitative RT-PCR, Expressing, Transfection, Staining, Activity Assay

Journal: Journal of Healthcare Engineering

Article Title: TSG Targeting KDM5A Affects Osteogenic Differentiation of Bone Mesenchymal Stem Cells Induced by Bone Morphogenetic Protein 2

doi: 10.1155/2022/6472864

Figure Lengend Snippet: TSG treatment inhibits the expression of KDM5A. (a) The expression level of KDM5A was detected by qPCR. (b) Western blot detection of KDM5A expression. Data were mean ± (S) D, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: Small interfering RNA (siRNA) targeting KDM5A (si-KMD5A) and siRNA negative control (si-NC) were all obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot

Journal: Journal of Healthcare Engineering

Article Title: TSG Targeting KDM5A Affects Osteogenic Differentiation of Bone Mesenchymal Stem Cells Induced by Bone Morphogenetic Protein 2

doi: 10.1155/2022/6472864

Figure Lengend Snippet: TSG promotes osteogenic differentiation of bone mesenchymal stem cells by targeting the expression of KDM5A. (a) qRT-PCR analysis of KDM5A expression in MSCs after different treatments. (b) Alkaline phosphatase activity was detected by ALP staining after osteogenic induction of MSCs in each group. (c) Alizarin Red S staining detection for mineralized nodules formed by bone marrow mesenchymal stem cells after staining detection in each group. (d) qPCR was used to detect the OPN expression of MSCs in each group after osteogenesis induction. (e) qPCR detection of OCN expression in MSCs treated in each group after osteogenesis induction. (f) qPCR was used to detect Runx2 expression in MSCs in each group after osteogenic induction. (g) qPCR was used to detect Osterix expression in MSCs treated in each group after osteogenic induction. (h) qPCR was used to detect the expression level of Col1a1 in MSCs in each group after osteogenic induction. MSC/V: cells treated with empty plasmid (vector). Data were mean ± (S) D, ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Small interfering RNA (siRNA) targeting KDM5A (si-KMD5A) and siRNA negative control (si-NC) were all obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Staining, Plasmid Preparation

Journal: bioRxiv

Article Title: The Epigenetic Regulator Histone Demethylase KDM5A is Activated and Pathogenic in a Mouse Model of Heart Failure

doi: 10.64898/2026.01.05.697798

Figure Lengend Snippet: (A) Immunoblot analysis of whole-heart lysates from Wild-type and Myh6-Cre:Lmna F/F mice showing KDM5A protein expression and normalization control GAPDH. (B) Quantification of KDM5A protein levels normalized to GAPDH, demonstrating increased expression in Myh6-Cre:Lmna F/F hearts compared to Wild-type (n=5 Wild-type and n=4 Myh6-Cre:Lmna F/F ). (C) Immunoblot analysis of isolated cardiomyocytes from Wild-type and Myh6-Cre:Lmna F/F mice showing KDM5A expression. (D) Quantification of KDM5A protein levels from isolated cardiomyocytes normalized to Vinculin (VCL), confirming increased expression in Myh6-Cre:Lmna F/F samples (n=7 Wild-type and Myh6-Cre:Lmna F/F ). (E) Immunoblot analysis of nuclear extracts from isolated cardiomyocytes of Wild-type and Myh6-Cre:Lmna F/F mice. (F) Quantification of nuclear KDM5A protein levels normalized to Histone H3, showing enhanced nuclear retention in Myh6-Cre:Lmna F/F cardiomyocytes (n=4 Wild-type and Myh6-Cre:Lmna F/F ). (G) Transcript levels of representative KDM5A target genes in cardiomyocytes, showing suppression of genes in Myh6-Cre:Lmna F/F samples as compared to Wild-type (n=4 Wild-type and Myh6-Cre:Lmna F/F ). All data are presented as mean ± 95% confidence interval (CI). Normality of data distribution was assessed using the Shapiro–Wilk test. Statistical significance was evaluated using the Student’s t -test for normally distributed variables (Panel B, D and G) or the Mann–Whitney U test for non-normally distributed variables (Panel F). The corresponding p -values are shown.

Article Snippet: Wild-type (C57BL/6J), Myh6-Cre (C57BL/6J), and Kdm5a floxed mice (JB6.129S6(Cg)-Kdm5a tm1Kael /J) were obtained from Jackson Laboratory.

Techniques: Western Blot, Expressing, Control, Isolation, MANN-WHITNEY

Journal: bioRxiv

Article Title: The Epigenetic Regulator Histone Demethylase KDM5A is Activated and Pathogenic in a Mouse Model of Heart Failure

doi: 10.64898/2026.01.05.697798

Figure Lengend Snippet: ( A-B) Immunoblot analysis of isolated cardiomyocytes from P16 mice, which confirmed the absence of KDM5A protein following cardiomyocyte-specific deletion, normalized to Vinculin (VCL) as a loading control (n=3). (B) Quantitative assessment of KDM5A deletion. Statistical analysis was performed by Kruskal-Wallis test and Dunn post hoc pairwise comparisons P values are shown. (C) Kaplan–Meier survival analysis showing that loss of Kdm5a in cardiomyocytes has no significant effect on overall survival compared with control groups. (D) Echocardiographic measurements obtained from four groups of mice assessing body weight, heart rate, left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), and fractional shortening (FS). No significant differences were observed across genotypes. Data are presented as mean ± 95% confidence interval (CI). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s post hoc pairwise comparisons or Kruskal-Wallis test and Dunn post hoc pairwise comparison. The one-way ANOVA or Kruskal-Wallis test P values are shown in the figure. (E) TUNEL staining of myocardial sections from 2-month-old mice (n = 4 per group) showing apoptotic nuclei (green) and DAPI (blue). (F) Quantification of TUNEL + nuclei expressed as percentage of total nuclei, showing no difference between the groups (n=4,mean ± SD, one-way ANOVA with post hoc Tukey pairwise comparison).

Article Snippet: Wild-type (C57BL/6J), Myh6-Cre (C57BL/6J), and Kdm5a floxed mice (JB6.129S6(Cg)-Kdm5a tm1Kael /J) were obtained from Jackson Laboratory.

Techniques: Western Blot, Isolation, Control, Comparison, TUNEL Assay, Staining

Journal: bioRxiv

Article Title: The Epigenetic Regulator Histone Demethylase KDM5A is Activated and Pathogenic in a Mouse Model of Heart Failure

doi: 10.64898/2026.01.05.697798

Figure Lengend Snippet: (A) Quantitative RT-PCR showing deletion of mRNA levels of Lmna and Kdm5a in the cardiac myocytes showing substantial reduction of Lmna in Myh6-Cre:Lmna F/F and Myh6-Cre:Lmna F/F :Kdm5a F/F and Kdm5a in the latter group compared to WT controls. (B-C) Immunoblot and quantitative analysis showing reduction of LMNA in the Myh6-Cre:Lmna F/F and Myh6-Cre:Lmna F/F :Kdm5a F/F and KDM5A in the Myh6-Cre:Lmna F/F :Kdm5a F/F group. The data confirmed deletion of both genes. (D) Body weight, heart rate, and selected echocardiographic indices were measured in Wild-type, Myh6-Cre:Lmna F/F and Myh6-Cre:Lmna F/F :Kdm5a F/F mice (n = 14 per group). Parameters include Body weight, LVESD (left ventricular end-systolic diameter), LVEDD (left ventricular end-diastolic diameter), LVEDDi (LVEDD indexed to body weight), FS (fractional shortening) and EF (ejection fraction). Statistical significance was determined by one-way ANOVA followed by Tukey post hoc test. (E) Kaplan–Meier survival curves demonstrating prolonged median and maximal survival in Myh6-Cre:Lmna F/F :Kdm5a F/F mice compared to Myh6-Cre:Lmna F/F . (F) Transcript levels of selected markers of cardiac dysfunction measured by quantitative RT-qPCR in isolated cardiomyocytes from P21 mice. Expression values were normalized to Gapdh . Data are presented as mean ± 95% confidence interval (CI). N= 6-7, Statistical significance was determined using one-way ANOVA followed by Tukey post hoc pairwise comparisons.

Article Snippet: Wild-type (C57BL/6J), Myh6-Cre (C57BL/6J), and Kdm5a floxed mice (JB6.129S6(Cg)-Kdm5a tm1Kael /J) were obtained from Jackson Laboratory.

Techniques: Quantitative RT-PCR, Western Blot, Isolation, Expressing

Journal: bioRxiv

Article Title: The Epigenetic Regulator Histone Demethylase KDM5A is Activated and Pathogenic in a Mouse Model of Heart Failure

doi: 10.64898/2026.01.05.697798

Figure Lengend Snippet: (A) Gene set enrichment analysis (GSEA) of RNA-seq data showing significant induction of apoptotic gene signatures in Myh6-Cre:Lmna F/F cardiomyocytes and reversal of this signature upon Kdm5a deletion in Myh6-Cre:Lmna F/F : Kdm5a F/F CM. (B) Representative TUNEL staining of myocardial sections from P21 mice demonstrating increased apoptotic nuclei (green) in Myh6-Cre:Lmna F/F hearts, counterstained with DAPI (blue). (C) Quantification of TUNEL + nuclei expressed as percentage of total nuclei, showing a marked increase in Myh6-Cre:Lmna F/F hearts compared with Wild-type and a significant reduction upon Kdm5a deletion. N = 6 per group. Statistical significance was determined using one-way ANOVA followed by Tukey post hoc pairwise comparisons. (D) Transcript levels of pro-apoptotic genes measured by quantitative RT-qPCR from whole heart RNA at P21, normalized to Gapdh , showing upregulation in Myh6-Cre:Lmna F/F and partial restoration in Myh6-Cre:Lmna F/F : Kdm5a F/F . N = 7 biological replicates per group were used and Statistical significance was determined using one-way ANOVA followed by Tukey post hoc pairwise comparisons. (E) Representative immunoblot (IB) analysis of cardiomyocyte extracts from P21 mice showing expression of CASP3, BAX, and BAD across the three groups. (F) Quantification of IB data (normalized to VCL) reveals increased expression of pro-apoptotic proteins in Myh6-Cre:Lmna F/F hearts, which are attenuated following Kdm5a deletion. N = 8 biological replicates per group were used and Statistical significance was determined using one-way ANOVA followed by Tukey post hoc pairwise comparisons.

Article Snippet: Wild-type (C57BL/6J), Myh6-Cre (C57BL/6J), and Kdm5a floxed mice (JB6.129S6(Cg)-Kdm5a tm1Kael /J) were obtained from Jackson Laboratory.

Techniques: RNA Sequencing, TUNEL Assay, Staining, Quantitative RT-PCR, Western Blot, Expressing

Journal: bioRxiv

Article Title: The Epigenetic Regulator Histone Demethylase KDM5A is Activated and Pathogenic in a Mouse Model of Heart Failure

doi: 10.64898/2026.01.05.697798

Figure Lengend Snippet: (A–B) Representative picrosirius red–stained myocardial sections from 3-week-old Wild-type, Myh6-Cre:Lmna F/F , and Myh6-Cre:Lmna F/F : Kdm5a F/F mice, with corresponding quantitative analysis of collagen volume fraction (CVF). CVF was markedly elevated in Myh6-Cre:Lmna F/F hearts (9.8 ± 3.8%) compared to Wild-type (~1%), and this increase was significantly attenuated in Myh6-Cre:Lmna F/F : Kdm5a F/F hearts (1.5 ± 0.85%). Collagen volume fraction was quantified using ImageJ. For each genotype, n=5 mice hearts were used and statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. (C) Transcript levels of fibrosis-associated genes in Wild-type, Myh6-Cre:Lmna F/F , and Myh6-Cre:Lmna F/F : Kdm5a F/F hearts, measured by quantitative RT-qPCR. N=7 biological replicates per group were used and Statistical significance was determined using one-way ANOVA followed by Tukey post hoc pairwise comparisons.

Article Snippet: Wild-type (C57BL/6J), Myh6-Cre (C57BL/6J), and Kdm5a floxed mice (JB6.129S6(Cg)-Kdm5a tm1Kael /J) were obtained from Jackson Laboratory.

Techniques: Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The Epigenetic Regulator Histone Demethylase KDM5A is Activated and Pathogenic in a Mouse Model of Heart Failure

doi: 10.64898/2026.01.05.697798

Figure Lengend Snippet: (A) Principal component analysis (PCA) of RNA-seq data from Wild-type (black), Myh6-Cre:Lmna F/F (red), and Myh6-Cre:Lmna F/F : Kdm5a F/F (green) CM showing distinct clustering of transcriptional profiles. Each dot represents an individual biological replicate. (B) Volcano plot of differentially expressed genes (DEGs) in Myh6-Cre:Lmna F/F vs. Wild-type hearts, highlighting significantly upregulated (red) and downregulated (blue) genes. (C) Volcano plot of DEGs in Myh6-Cre:Lmna F/F : Kdm5a F/F vs. Myh6-Cre:Lmna F/F CM, showing transcriptional rescue upon Kdm5a deletion. (D) The Venn diagram depicting the overlap of DEGs showing rescue after Kdm5a loss and heatmap of rescue gene showing widespread transcriptional alterations in Myh6-Cre:Lmna F/F compared to Wild-type, with partial restoration of expression patterns following Kdm5a deletion. (E) Gene Set Enrichment Analysis (GSEA) plots for KDM5A target genes. Left: Myh6-Cre:Lmna F/F vs. Wild-type, showing significant enrichment of KDM5A targets. Right: Myh6-Cre:Lmna F/F : Kdm5a F/F vs. Myh6-Cre:Lmna F/F , showing reversal of KDM5A-driven transcriptional repression. (F) Hallmark pathway enrichment analysis highlighting rescue of oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and myogenesis pathways upon Kdm5a deletion. (G) Downregulated hallmark pathways in Myh6-Cre:Lmna F/F : Kdm5a F/F relative to Myh6-Cre:Lmna F/F , including TNFα signalling, hypoxia, EMT, and p53 pathways. (H) IPA predicted upstream regulators that show inhibition in Myh6-Cre:Lmna F/F : Kdm5a F/F compared to Myh6-Cre:Lmna F/F , including KDM5A, TP53, and HIF1A. (I) IPA predicted activated upstream regulators in Myh6-Cre:Lmna F/F : Kdm5a F/F compared to Myh6-Cre:Lmna F/F , including ESRRA, and PGC1A.

Article Snippet: Wild-type (C57BL/6J), Myh6-Cre (C57BL/6J), and Kdm5a floxed mice (JB6.129S6(Cg)-Kdm5a tm1Kael /J) were obtained from Jackson Laboratory.

Techniques: RNA Sequencing, Expressing, Phospho-proteomics, Inhibition

Journal: bioRxiv

Article Title: The Epigenetic Regulator Histone Demethylase KDM5A is Activated and Pathogenic in a Mouse Model of Heart Failure

doi: 10.64898/2026.01.05.697798

Figure Lengend Snippet: (A) Total number of H3K4me3 peaks identified by CUT&RUN in Wild-type (black), Myh6-Cre:Lmna F/F (red), and Myh6-Cre:Lmna F/F : Kdm5a F/F (green) CM. Each dot represents an individual biological replicate; bars indicate mean ± SD. (B) Genomic annotation of H3K4me3 peaks across genotypes. (C) Mean H3K4me3 peak length per sample, showing similar distribution across genotypes. (D) Aggregate H3K4me3 signal profiles centred on genome wide transcription start sites (TSS ±5 kb), demonstrating promoter-proximal H3K4me3 enrichment. (E) Principal component analysis (PCA) of genome-wide H3K4me3 profiles separates WT, Myh6-Cre:Lmna F/F , and Myh6-Cre:Lmna F/F : Kdm5a F/F samples, indicating distinct chromatin states and partial normalization following Kdm5a loss. (F) Volcano plot showing differentially enriched H3K4me3 peaks in Myh6-Cre:Lmna F/F relative to Wild-type. Red, enriched; blue, depleted; grey, not significant. (G) Volcano plot of H3K4me3 changes in Myh6-Cre:Lmna F/F : Kdm5a F/F relative to Myh6-Cre:Lmna F/F , showing restoration of promoter-associated H3K4me3 peaks. (H) Average profiles and heatmaps of H3K4me3 signal at peaks enriched in Myh6-Cre:Lmna F/F : Kdm5a F/F relative to Myh6-Cre:Lmna F/F , demonstrating recovery of promoter H3K4me3 occupancy toward Wild-type levels. (I) Gene ontology and pathway enrichment analysis of genes associated with rescued H3K4me3 peaks, revealing restoration of muscle contraction, cardiac conduction, metabolic, and nuclear receptor signalling programs. (J) Average profiles and heatmaps of H3K4me3 signal at peaks depleted in Myh6-Cre:Lmna F/F : Kdm5a F/F relative to Myh6-Cre:Lmna F/F , indicating attenuation of aberrantly elevated promoter marks. (K) Pathway enrichment analysis of genes associated with depleted peaks, highlighting downregulation of RHO GTPase signalling, extracellular matrix organization, and VEGF-associated pathways.

Article Snippet: Wild-type (C57BL/6J), Myh6-Cre (C57BL/6J), and Kdm5a floxed mice (JB6.129S6(Cg)-Kdm5a tm1Kael /J) were obtained from Jackson Laboratory.

Techniques: Genome Wide

Journal: bioRxiv

Article Title: The Epigenetic Regulator Histone Demethylase KDM5A is Activated and Pathogenic in a Mouse Model of Heart Failure

doi: 10.64898/2026.01.05.697798

Figure Lengend Snippet: (A) Representative IGV genome browser tracks showing H3K4me3 enrichment at cardiac transcription factor Tbx5 cis regulatory elements and promoter region. Tracks shown on mm39 genome assembly. Signals from WT (black), Myh6-Cre:Lmna F /F (red), and Myh6-Cre:Lmna F /F : Kdm5a F/F (green) hearts are displayed. (B) Quantitative RT-PCR analysis of Tbx5 transcript levels across genotypes (n = 7 per group), demonstrating suppression in Myh6-Cre:Lmna F /F and rescue upon Kdm5a deletion. (C) Quantitative RT-PCR analysis of downstream targets of TBX5 (n = 7 per group), demonstrating restoration of expression in Myh6-Cre:Lmna F /F : Kdm5a F/F . (D) Representative IGV genome browser tracks showing H3K4me3 enrichment at transcription factor Esrrg cis regulatory and promoter region. Signals from Wild-type (black), Myh6-Cre:Lmna F /F (red), and Myh6-Cre:Lmna F /F : Kdm5a F/F (green) hearts are displayed. (E) Quantitative RT-PCR analysis of Esrrg transcript levels across genotypes (n = 7 per group), demonstrating suppression in Myh6-Cre:Lmna F /F and rescue upon Kdm5a deletion. (F-G) Quantitative RT-PCR analysis of ESRRG target genes encoding OXPHOS complex ( Cox7a1, Cox7a2, Ndufa6, Ndufa3, Sdhb, Cox10, Ndufs2, Uqcr2, Uqcr10 ) and fatty acid oxidation regulators ( Cpt1b, Acadvl, Acadm, Echdc3 ) in Myh6-Cre:Lmna F /F , all of which were significantly restored upon Kdm5a deletion (n=6-7 per group). Gene set enrichment analysis demonstrating overall suppression of fatty acid metabolism and OXPHOS in Myh6-Cre:Lmna F /F compared to Wild-type and partially restored in Myh6-Cre:Lmna F /F : Kdm5a F/F . Statistical significance was determined by one-way ANOVA and Tukey post hoc pairwise comparisons.

Article Snippet: Wild-type (C57BL/6J), Myh6-Cre (C57BL/6J), and Kdm5a floxed mice (JB6.129S6(Cg)-Kdm5a tm1Kael /J) were obtained from Jackson Laboratory.

Techniques: Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Differences in substrate engagement and Retinoblastoma protein (RB) binding of human KDM5A and KDM5B

doi: 10.64898/2026.04.30.721888

Figure Lengend Snippet: B. (A) Top: AlphaFold3 prediction of KDM5B. Regions predicted with low confidence and high disorder content are not shown for clarity (aa234-309; aa1284-1344; aa1370-1562; for the prediction metrics see Suppl. Fig. 3). Bottom: Domain architecture of KDM5 proteins according to Uniprot database annotation. Green, catalytic JmjN/C domain; blue, ARID domain; orange, PHD1-3 and C5HC2 zinc finger domains; red, reported RB interaction sites. (B,C) Negative-stain EM 3D reconstructions (top) and representative 2D class averages (bottom) of KDM5A (B) and KDM5B (C). AlphaFold3 predictions were fitted into the densities by rigid-body docking. The AlphaFold3 models were separated after aa797 for KDM5A and after aa755 for KDM5B to accommodate the orientation of the C-terminal coiled-coil region. Scale bars = 24 nm. (D) Electrophoretic mobility shift assay (EMSA) of KDM5A and KDM5B with 100 nM H3K4me3MLA nucleosomes (nucl). DNA stained with SybrGold. BP: base pairs (marker). Image representative of five independent experiments (E-G). FDH-based demethylase activity assays with H3 1-18 K4me3 peptides. Michaelis–Menten plot (F) and respective calculated kinetic rate in min -1 (G) and K M in µM (H) of WT KDM5A (green), KDM5A Δ LxCxE (red), KDM5A active core (KDM5A 1-797), (purple), WT KDM5B (blue), KDM5B LxCxE insertion, and KDM5B active core (KDM5B1-755). Means of at least three independent experiments and the standard error of the mean (SEM) are shown. Significance was determined using one-way ANOVA, with P < 0.05 considered significant. P values below 0.01 are highlighted with two asterisks and below 0.0001 with four.

Article Snippet: Codon-optimized coding sequences of KDM5A/B (N-terminally StrepII-tagged) were purchased from Twist Bioscience and cloned into GoldenBac vectors (pGB [ ] ) for insect cell expression.

Techniques: Staining, Electrophoretic Mobility Shift Assay, Marker, Activity Assay

Journal: Nature

Article Title: An extra-erythrocyte role of haemoglobin body in chondrocyte hypoxia adaption

doi: 10.1038/s41586-023-06611-6

Figure Lengend Snippet: a , b , Expression of the indicated genes in primary cultured chondrocytes of mouse fetal growth plates upon conditional knockout of Hif-1α in either 20% O 2 ( a ) or 1% O 2 for 6 h ( b ). n = 3 biologically independent samples. c , d , Expression of the indicated genes in primary cultured chondrocytes of mouse fetal growth plates upon conditional knockout of Hif-2α in either 20% O 2 ( c ) or 1% O 2 for 6 h ( d ). n = 3 biologically independent samples. e , f , Expression of the indicated genes in primary cultured chondrocytes of mouse fetal growth plates upon conditional knockout of both Hif-1α and Hif-2α ( Hif-1/2α ) in either 20% O 2 ( e ) or 1% O 2 for 6 h ( f ). n = 3 biologically independent samples. g , Quantification of Kdm5a protein by Western blot analysis of total protein lysate extracted from Ctrl or si- Kdm5a primary cultured chondrocytes. A representative Western blot is shown on the left, and quantification of all biological replicates is provided on the right. Data were normalized to α-tubulin (n = 3 biologically independent experiments). For gel source data, see Supplementary Fig. . h , Quantification of Kdm5b protein by Western blot analysis of total protein lysate extracted from Ctrl or si- Kdm5b primary cultured chondrocytes. A representative Western blot is shown on the left, and quantification of all biological replicates is provided on the right. Data were normalized to α-tubulin (n = 3 biologically independent experiments). For gel source data, see Supplementary Fig. . i , j , Expression of the indicated genes in primary cultured chondrocytes upon knockdown of Kdm5a or Kdm5b in either 20% ( i ) or 1% O 2 ( j ) for 6 h. n = 3 biologically independent experiments. k , l , ChIP-qPCR analysis results of H3K4me3 for the indicated genes in primary cultured chondrocytes siRNA depleted of Kdm5a in either 20% ( k ) or 1% O 2 ( l ). The data are mean with SEM of triplicate experiments. P values were calculated using two-tailed Student’s t -test ( a - f ) or one-way ANOVA tests ( g - l ). The exact P -values of comparison are presented in the figures, respectively.

Article Snippet: After gel transfer to PVDF membranes using a Bio-Rad Criterion system, blots were blocked in 5% non-fat milk/1× TBST for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: HIF1α (20960-1-AP, Proteintech) at 1:1,000, HIF2α (also known as Epas1) (NB100-122, Novus) at 1:1,000, KLF1 (also known as EKLF) (PAB5859, Abnova) at 1:1,000, AMPK (A1229, ABclonal) at 1:1000, pAMPK (AP1002, ABclonal) at 1:1,000, caspase 3 (A19654, ABclonal) at 1:1,000, KDM5A (A4755, ABclonal) at 1:1,000, and KDM5B (A15740, ABclonal) at 1:1,000.

Techniques: Expressing, Cell Culture, Knock-Out, Western Blot, Two Tailed Test, Comparison