Journal: Cell Death and Differentiation

Article Title: Inhibition of EZH2 induces NK cell-mediated differentiation and death in muscle-invasive bladder cancer

doi: 10.1038/s41418-019-0278-9

Figure Lengend Snippet: Mutations in KDM6A and SWI/SNF complex genes are common in muscle-invasive bladder cancer. a Exome-sequencing analysis of 138 bladder tumor patients from Roswell Park Comprehensive Cancer Center (Roswell Park) revealed mutations in several chromatin remodeling enzymes. KMD6A, a histone demethylase, responsible for removing trimethylation mark on H3K27 is mutated in 20% while SWI/SNF complex family members are mutated in 18% of tumors. Mutations include missense and nonsense mutations as well as frameshift deletions, insertions and in frame deletions. b RNA-sequencing of a 66 tumors revealed significant alterations in gene expression of EZH2 and members of the SWI/SNF complex. c z -scores of PRC2-regulated genes were lower in tumors with KDM6A and KDM6A/SWI/SNF mutations compared to tumors without any mutations or SWI/SNF mutations alone

Article Snippet: Primary antibodies used were: H3K27me3 (Cell signaling, 9733S), total histone H3 (Cell Signaling, 9715), EZH2 (Cell signaling, 5246), KDM6A (Atlas Antibodies, HPA002111), ALDH2 (Abcam, ab108306), and CK5 (Covance, PRB 160-P).

Techniques: Sequencing, RNA Sequencing, Gene Expression

Journal: Cell Death and Differentiation

Article Title: Inhibition of EZH2 induces NK cell-mediated differentiation and death in muscle-invasive bladder cancer

doi: 10.1038/s41418-019-0278-9

Figure Lengend Snippet: HT1376 xenografts with KDM6A and ARID1A mutations are sensitive to EZH2 inhibition alone and in combination with cisplatin. a 500 mg/kg EPZ011989 alone or in combination with 3 mg/ml cisplatin reduced HT1376 tumor volume in nude mice. The Y -axis represents tumor volume in mm 3 and the X -axis represents number of days in the study. There is a significant difference in the tumor growth rate between the combination treatment and cisplatin alone treatment. b Mathematical modeling of tumor growth rate indicated that the vehicle, Cisplatin, EPZ011989 would grow tumors from 100 to 1000 mm 3 in 58.5, 77.7, and 399.8 days, respectively. The combination treatment is predicted to not reach 1000 mm 3 . c End-point tumor weights showed that the combination treatment had significantly lower tumor weight as compared cisplatin alone treatment. The Y -axis represents the tumor weight in grams and the X -axis represents the different groups. Error bars represent standard error of means of all the mice in each group. One-way ANOVA model with pairwise comparisons made as appropriate were performed to test differences between single and combination treatments. * p < 0.05, ** p < 0.01, *** p < 0.001, triangle indicates significant difference as compared to vehicle, hash indicates significant difference between the EPZ011989 and cisplatin alone treatments

Article Snippet: Primary antibodies used were: H3K27me3 (Cell signaling, 9733S), total histone H3 (Cell Signaling, 9715), EZH2 (Cell signaling, 5246), KDM6A (Atlas Antibodies, HPA002111), ALDH2 (Abcam, ab108306), and CK5 (Covance, PRB 160-P).

Techniques: Inhibition

Journal: Cell Death and Differentiation

Article Title: Inhibition of EZH2 induces NK cell-mediated differentiation and death in muscle-invasive bladder cancer

doi: 10.1038/s41418-019-0278-9

Figure Lengend Snippet: EZH2 inhibition is associated with NK cell activation. a Gene set enrichment analysis shows that EZH2 inhibition alone or in combination with cisplatin is associated with increased in IFN-γ signaling. qPCR analysis showed that NK cell activity associated transcripts IFN-γ , MIP-1α , CD3D , CYTH1 , CD86 , ICAM1 , and ICAM2 were significantly upregulated in the combination treatment as compared to cisplatin alone treatment. Error bars represent standard error of means of all the mice in each group. One-way ANOVA model with pairwise comparisons made as appropriate were performed to test differences between single and combination treatments. * p < 0.05, ** p < 0.01, triangle indicates significant difference as compared to vehicle, hash indicates significant difference between the EPZ011989 and cisplatin alone treatments. b , c CD56, an NK cell-associated marker is significantly higher in combination-treated tumors compared to single agent and vehicle-treated mice. NCR1, Natural Cytotoxic triggering Receptor 1, that mediates tumor cell lysis is significantly increased in the EPZ011989 and combination-treated groups compared to vehicle and cisplatin-treated groups. Orange arrows indicate infiltrating cells and black arrows point to tumor cells. Error bars represent standard error of means of Image J quantitation in all the mice in each group. One-way ANOVA model with pairwise comparisons made as appropriate were performed to test differences between single and combination treatments. * p < 0.05, ** p < 0.01, triangle indicates significant difference as compared to vehicle, hash indicates significant difference between the EPZ011989 and cisplatin alone treatments. d RNA-sequencing of a 66 tumors revealed significant alterations in gene expression of a subset of ligands and receptors that are involved in the activation of natural killer (NK) cells. e z -scores of NK related transcripts are higher in tumors with no mutations compared to tumors with KDM6A and/or SWI/SNF mutations

Article Snippet: Primary antibodies used were: H3K27me3 (Cell signaling, 9733S), total histone H3 (Cell Signaling, 9715), EZH2 (Cell signaling, 5246), KDM6A (Atlas Antibodies, HPA002111), ALDH2 (Abcam, ab108306), and CK5 (Covance, PRB 160-P).

Techniques: Inhibition, Activation Assay, Activity Assay, Marker, Lysis, Quantitation Assay, RNA Sequencing, Gene Expression

Journal: Cell Death and Differentiation

Article Title: Inhibition of EZH2 induces NK cell-mediated differentiation and death in muscle-invasive bladder cancer

doi: 10.1038/s41418-019-0278-9

Figure Lengend Snippet: EZH2 inhibition-mediated NK cell activation reprograms bladder tumor cells by reducing expression of markers associated with pluripotent potential. a , b Markers of pluripotency in bladder cancer, CK5, ALDH2, and p63, were stained heterogeneously in all tumors. CK5 expression was significantly reduced and ALDH2 was reduced in EPZ011989 and combination-treated tumors compared with vehicle and cisplatin-treated tumors. p63 expression was significantly higher in cisplatin-treated tumors compared to vehicle group. Orange arrows indicate infiltrating cells and white arrows point to tumor cells. c RNA-sequencing analysis of Roswell Park muscle-invasive bladder tumors reveal significant upregulation of markers of cells with pluripotency potential, with the exception of ALDH2, compared to non-tumor tissues. d Pluripotency-associated markers were significantly lower in tumors with KDM6A mutation compared to all the mutation subtypes. e EZH2-mediated H3K27me3 deposition on histones is opposed by wild-type KDM6A and SWI/SNF complex. Tumor cells exhibit greater pluripotency potential and decreased NK cell activity when KDM6A and SWI/SNF loss of function mutations are unable to counteract EZH2 activity. EZH2 inhibition in this context increases NK cell activity measured by IFN-γ that mediates decrease in CK5 and ALDH2 that are pluripotency-associated markers. Greater NK cell activity and reduced pluripotency further augments cisplatin-based chemotherapy in muscle-invasive bladder cancer

Article Snippet: Primary antibodies used were: H3K27me3 (Cell signaling, 9733S), total histone H3 (Cell Signaling, 9715), EZH2 (Cell signaling, 5246), KDM6A (Atlas Antibodies, HPA002111), ALDH2 (Abcam, ab108306), and CK5 (Covance, PRB 160-P).

Techniques: Inhibition, Activation Assay, Expressing, Staining, RNA Sequencing, Mutagenesis, Activity Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: A methyl-to-acetyl switch in H3K27 drives metabolic reprogramming and resistance to BRAF V600E inhibition in melanoma

doi: 10.1016/j.neo.2025.101223

Figure Lengend Snippet: Inhibition of KDM6A prevents the H3K27 methyl-to-acetyl switch and gene activation induced by BRAF V600E inhibition. (A) Pharmacological inhibition of KDM6A by GSKJ4 in A375P cells increases H3K27me3 while reducing H3K27ac in a dose-dependent manner. Cells were treated with DMSO or GSKJ4 at the indicated concentration for 24 h. (B-C) GSKJ4 treatment inhibits the BRAF V600E inhibition–induced changes in H3K27me3 and H3K27ac in A375P cells, as shown by Western blot ( B ) and immunofluorescence ( C ). Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 24 h. (D) In A375P cells, KDM6A inhibition blunts the PLX4032-mediated alterations in H3K27ac occupancy at the PGC1α, DCT, MET, and WDR19 promoters, as determined by ChIP. Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 16 h. (E) GSKJ4 treatment abrogates the upregulation of EZH2 target genes (PGC1α, DCT, MET, WDR19) by BRAF V600E inhibition in A375P cells. Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 16 h. ( F ) KDM6A knockout efficiency in A375P cells was confirmed by qRT-PCR and Western blot. (G-I) CRISPR-mediated KDM6A deletion prevents the changes in H3K27me3 and H3K27ac triggered by BRAF V600E inhibition in A375P cells, as shown by Western blot ( G ) and immunofluorescence ( H and I ). Cells were treated with DMSO or 2 μM PLX4032 for 24 h. (J) KDM6A deletion abolishes the alterations in H3K27ac occupancy at the PGC1α, DCT, MET, and WDR19 promoters in A375P cells. Cells were treated with DMSO or 2 μM PLX4032 for 16 h. (K) CRISPR-mediated KDM6A deletion suppresses the BRAF V600E inhibition–induced upregulation of EZH2 target genes in A375P cells. Cells were treated with DMSO or 2 μM PLX4032 for 16 h. Data are presented as mean ± SEM and representative of two to three independent experiments with similar results ( n = 3/group/experiment). * P < 0.05, ** P < 0.01, *** P < 0.001 by Student’s t-test in (F) and by two-way ANOVA in (C), (D), (E), (I), (J), and (K).

Article Snippet: Antibodies included: KDM6A (NOVUS, #NBP1-80628), BRD4 (HUABIO, #HA722785), HRP-conjugated β-Actin (ABclonal, #AC043), EZH2 (D2C9) (Cell Signaling Technology, #5246S), Histone H3 (D1H2) (CST, #4499S), H3K27me3 (C36B11) (CST, #9733S), H3K27ac (D5E4) (CST, #8173S), Cleaved PARP (HUABIO, #ET1608-10), Alpha Tubulin (HUABIO, # ER130905 ), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (CST, #4370S), and p44/42 MAPK (Erk1/2) (137F5) (CST, #4695).

Techniques: Inhibition, Activation Assay, Concentration Assay, Western Blot, Immunofluorescence, Knock-Out, Quantitative RT-PCR, CRISPR

Journal: Neoplasia (New York, N.Y.)

Article Title: A methyl-to-acetyl switch in H3K27 drives metabolic reprogramming and resistance to BRAF V600E inhibition in melanoma

doi: 10.1016/j.neo.2025.101223

Figure Lengend Snippet: Blockade of the KDM6A–H3K27ac–BRD4 axis reprograms melanoma metabolism. (A) Pharmacological inhibition of BRD4 by JQ1 attenuates the PLX4032-induced mitochondrial metabolic program in A375P cells. Cells were treated with DMSO, 2 μM JQ1, 2 μM PLX4032, or both for 16 h. (B) CRISPR-mediated BRD4 depletion suppresses the mitochondrial metabolic program triggered by PLX4032 in A375P melanoma cells. Cells were treated with DMSO or 2 μM PLX4032 for 16 h. (C-D) BRD4 inactivation reduces mitochondrial content ( C ) and respiration ( D ) induced by BRAF V600E inhibition in A375P cells, as measured by MitoTracker staining and a Clark-type oxygen electrode, respectively. Cells were treated with DMSO, 2 μM JQ1, 2 μM PLX4032, or both for 24 h. (E) Pharmacological inhibition of KDM6A activity by GSKJ4 prevents the PLX4032-mediated increase in the mitochondrial metabolic program in A375P cells. Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 16 h. (F) CRISPR-mediated KDM6A depletion suppresses the PLX4032-induced mitochondrial metabolic program in A375P melanoma cells. Cells were treated with DMSO or 2 μM PLX4032 for 16 h. (G-H) KDM6A inhibition decreases mitochondrial content ( G ) and respiration ( H ) following BRAF V600E inhibition in A375P cells. Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 24 h. Data are represented as mean ± SEM, and representative of two to three independent experiments with similar results ( n = 3/group/repeat). * P < 0.05, ** P < 0.01, and *** P < 0.001 by two-way ANOVA.

Article Snippet: Antibodies included: KDM6A (NOVUS, #NBP1-80628), BRD4 (HUABIO, #HA722785), HRP-conjugated β-Actin (ABclonal, #AC043), EZH2 (D2C9) (Cell Signaling Technology, #5246S), Histone H3 (D1H2) (CST, #4499S), H3K27me3 (C36B11) (CST, #9733S), H3K27ac (D5E4) (CST, #8173S), Cleaved PARP (HUABIO, #ET1608-10), Alpha Tubulin (HUABIO, # ER130905 ), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (CST, #4370S), and p44/42 MAPK (Erk1/2) (137F5) (CST, #4695).

Techniques: Inhibition, CRISPR, Staining, Activity Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: A methyl-to-acetyl switch in H3K27 drives metabolic reprogramming and resistance to BRAF V600E inhibition in melanoma

doi: 10.1016/j.neo.2025.101223

Figure Lengend Snippet: The H3K27 methyl-to-acetyl switch enables melanoma to survive BRAF V600E inhibition. (A) Basal expression levels of BRD4 or KDM6s negatively correlate with BRAF V600E inhibitor sensitivity across a panel of human melanoma cell lines. (B) In melanoma patients, basal BRD4 and KDM6s expression in pre-treatment samples negatively correlates with tumor response to BRAF V600E /MEK inhibitors. (C-D) CRISPR-mediated BRD4 depletion reduces melanoma cell survival ( C ) by increasing apoptotic cell death ( D ) following BRAF V600E inhibition. Cells were treated with DMSO or 2 μM PLX4032 for 24 h. (E-F) CRISPR-mediated KDM6A depletion similarly decreases melanoma survival ( E ) and enhances apoptotic cell death ( F ) in response to BRAF V600E inhibition. Cells were treated with DMSO or 2 μM PLX4032 for 24 h. (G) CRISPR-mediated KDM6A depletion in BRAF V600E -mutant YUMM1.7 melanoma cells significantly improves the in vivo response to BRAF V600E inhibition. The diagram illustrates the treatment strategy. The following group sizes were used: sgSCR_Vehicle ( n = 7), sgSCR_PLX4032 ( n = 8), sgKdm6a_Vehicle ( n = 6), and sgKdm6a_PLX4032 ( n = 9). In (A) and (B), each data point represents individual samples. In all other panels, data are presented as mean ± SEM, and are representative of two to three independent experiments with similar results in (C) and (E) ( n = 3/group/repeat). Statistical significance was determined by Pearson correlation in (A) and (B), or by two-way ANOVA with * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Antibodies included: KDM6A (NOVUS, #NBP1-80628), BRD4 (HUABIO, #HA722785), HRP-conjugated β-Actin (ABclonal, #AC043), EZH2 (D2C9) (Cell Signaling Technology, #5246S), Histone H3 (D1H2) (CST, #4499S), H3K27me3 (C36B11) (CST, #9733S), H3K27ac (D5E4) (CST, #8173S), Cleaved PARP (HUABIO, #ET1608-10), Alpha Tubulin (HUABIO, # ER130905 ), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (CST, #4370S), and p44/42 MAPK (Erk1/2) (137F5) (CST, #4695).

Techniques: Inhibition, Expressing, CRISPR, Mutagenesis, In Vivo

Journal: Neoplasia (New York, N.Y.)

Article Title: A methyl-to-acetyl switch in H3K27 drives metabolic reprogramming and resistance to BRAF V600E inhibition in melanoma

doi: 10.1016/j.neo.2025.101223

Figure Lengend Snippet: Targeting the H3K27 methyl-to-acetyl switch sensitizes melanoma to BRAF V600E inhibition. (A-B) BRD4 inactivation by JQ1 enhances the efficacy of PLX4032 in suppressing melanoma cell survival, as assessed by cell proliferation ( A ) and apoptosis (indicated by cleaved PARP1) ( B ). Cells were treated with DMSO, 2 μM JQ1, 2 μM PLX4032, or both for 24 h. (C-D) Pharmacological inhibition of KDM6A with GSKJ4 synergizes with PLX4032 to further suppress melanoma cell survival ( C ) and induce apoptotic cell death ( D ). Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 24 h. (E) The BRD4 inhibitor JQ1 significantly sensitizes BRAF V600E -mutant YUMM1.7 melanoma to PLX4032 in vivo . Vehicle ( n = 6), PLX4032 ( n = 5), JQ1 ( n = 5), PLX4032+JQ1 ( n = 10). The diagram illustrates the treatment strategy. (F) Inhibition of KDM6A by GSKJ4 significantly sensitizes BRAF V600E -mutant YUMM1.7 melanoma to PLX4032 in vivo . Vehicle ( n = 4), PLX4032 ( n = 4), GSKJ4 ( n = 4), PLX4032+GSKJ4 ( n = 5). (G-H) Treatment with GSKJ4 in combination with BRAF V600E inhibition reduces tumor progression ( G ) and prolongs survival ( H ) in a genetically engineered melanoma mouse model (Tyr::Cre ERT2 ;Braf CA ;Pten lox/lox ). Mice were induced with 4-hydroxytamoxifen (4-OHT; n = 5 per group for G, n = 6 per group for H). (I ) Diagram summarizing the interplay between BRAF V600E and the H3K27 epigenetic program in melanoma. Data are represented as mean ± SEM, and representative of two to three independent experiments with similar results in (A) and (C) ( n = 3/group/repeat). Statistical significance was determined by two-way ANOVA. Survival curves were estimated using the Kaplan–Meier method and compared using the log-rank (Mantel–Cox) test. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Antibodies included: KDM6A (NOVUS, #NBP1-80628), BRD4 (HUABIO, #HA722785), HRP-conjugated β-Actin (ABclonal, #AC043), EZH2 (D2C9) (Cell Signaling Technology, #5246S), Histone H3 (D1H2) (CST, #4499S), H3K27me3 (C36B11) (CST, #9733S), H3K27ac (D5E4) (CST, #8173S), Cleaved PARP (HUABIO, #ET1608-10), Alpha Tubulin (HUABIO, # ER130905 ), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (CST, #4370S), and p44/42 MAPK (Erk1/2) (137F5) (CST, #4695).

Techniques: Inhibition, Mutagenesis, In Vivo

Journal: Experimental and Therapeutic Medicine

Article Title: KDM6A suppresses hepatocellular carcinoma cell proliferation by negatively regulating the TGF-β/SMAD signaling pathway

doi: 10.3892/etm.2020.9000

Figure Lengend Snippet: Primer and shRNA sequences.

Article Snippet: Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-KDM6A (1:1,000; cat. no. orb333886; Biorbyt Ltd.), anti-transforming growth factor (TGF)-β (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-smad2 (1:1,000; cat. no. 18338; Cell Signaling Technology, Inc.), anti-Smad2 (1:1,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), anti-p-smad4 (1:1,000; cat. no. 10231-8-AP; ProteinTech Group, Inc.), anti-smad4 (1:1,000; cat. no. 10231-1-AP; ProteinTech Group, Inc.), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 10205-2-AP; ProteinTech Group, Inc.), anti-Ki67 (1:1,000; cat. no. 27309-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) and anti-Flag (1:4,000; cat. no. F7425; Sigma-Aldrich; Merck KGaA).

Techniques: shRNA, Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: KDM6A suppresses hepatocellular carcinoma cell proliferation by negatively regulating the TGF-β/SMAD signaling pathway

doi: 10.3892/etm.2020.9000

Figure Lengend Snippet: KDM6A expression is decreased in HCC clinical samples and cell lines. (A) KDM6A mRNA expression levels in 30 paired tumor and matched normal hepatic tissues. (B) KDM6A expression levels in tumor and matched normal hepatic tissue samples obtained from two patients as determined by immunohistochemistry. Magnification, x400. KDM6A protein expression levels in six HCC cell lines (YY-8103, SNU-398, MHCC97-L, LM3 and Huh7) were (C) determined by western blotting and (D) semi-quantified. KDM6A, lysine demethylase 6A; HCC, hepatocellular carcinoma.

Article Snippet: Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-KDM6A (1:1,000; cat. no. orb333886; Biorbyt Ltd.), anti-transforming growth factor (TGF)-β (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-smad2 (1:1,000; cat. no. 18338; Cell Signaling Technology, Inc.), anti-Smad2 (1:1,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), anti-p-smad4 (1:1,000; cat. no. 10231-8-AP; ProteinTech Group, Inc.), anti-smad4 (1:1,000; cat. no. 10231-1-AP; ProteinTech Group, Inc.), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 10205-2-AP; ProteinTech Group, Inc.), anti-Ki67 (1:1,000; cat. no. 27309-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) and anti-Flag (1:4,000; cat. no. F7425; Sigma-Aldrich; Merck KGaA).

Techniques: Expressing, Immunohistochemistry, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: KDM6A suppresses hepatocellular carcinoma cell proliferation by negatively regulating the TGF-β/SMAD signaling pathway

doi: 10.3892/etm.2020.9000

Figure Lengend Snippet: KDM6A overexpression inhibits hepatocellular carcinoma cell proliferation. (A) Transfection efficiency of KDM6A overexpression in Huh7 and LM3 cells. The effect of KDM6A overexpression on (B) Huh7 and (C) LM3 cell proliferation was measured by performing an MTT assay. (D) The effect of KDM6A overexpression on Huh7 and LM3 cell proliferation was detected by performing a crystal violet assay. Magnification, x4. (E) The OD value of the crystal violet assay in Huh7 and LM3 cells. ** P<0.01 and *** P<0.001 vs. vector. KDM6A, lysine demethylase 6A; OD, optical density.

Article Snippet: Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-KDM6A (1:1,000; cat. no. orb333886; Biorbyt Ltd.), anti-transforming growth factor (TGF)-β (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-smad2 (1:1,000; cat. no. 18338; Cell Signaling Technology, Inc.), anti-Smad2 (1:1,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), anti-p-smad4 (1:1,000; cat. no. 10231-8-AP; ProteinTech Group, Inc.), anti-smad4 (1:1,000; cat. no. 10231-1-AP; ProteinTech Group, Inc.), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 10205-2-AP; ProteinTech Group, Inc.), anti-Ki67 (1:1,000; cat. no. 27309-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) and anti-Flag (1:4,000; cat. no. F7425; Sigma-Aldrich; Merck KGaA).

Techniques: Over Expression, Transfection, MTT Assay, Crystal Violet Assay, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: KDM6A suppresses hepatocellular carcinoma cell proliferation by negatively regulating the TGF-β/SMAD signaling pathway

doi: 10.3892/etm.2020.9000

Figure Lengend Snippet: KDM6A knockdown promotes hepatocellular carcinoma cell proliferation. (A) Transfection efficiency of KDM6A knockdown in YY-8103 and SNU-398 cells. (B) The effect of KDM6A knockdown on (B) YY-8103 and (C) SNU-398 cell proliferation was measured by performing an MTT assay. (D) The effect of KDM6A knockdown on YY-8103 and SNU-398 cell proliferation was detected by performing a crystal violet assay. Magnification, x4. (E) The OD value of the crystal violet assay in YY-8103 and SNU-398 cells. *** P<0.001 vs. SCR. KDM6A, lysine demethylase 6A; OD, optical density; SCR, scrambled; sh, short hairpin RNA.

Article Snippet: Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-KDM6A (1:1,000; cat. no. orb333886; Biorbyt Ltd.), anti-transforming growth factor (TGF)-β (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-smad2 (1:1,000; cat. no. 18338; Cell Signaling Technology, Inc.), anti-Smad2 (1:1,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), anti-p-smad4 (1:1,000; cat. no. 10231-8-AP; ProteinTech Group, Inc.), anti-smad4 (1:1,000; cat. no. 10231-1-AP; ProteinTech Group, Inc.), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 10205-2-AP; ProteinTech Group, Inc.), anti-Ki67 (1:1,000; cat. no. 27309-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) and anti-Flag (1:4,000; cat. no. F7425; Sigma-Aldrich; Merck KGaA).

Techniques: Transfection, MTT Assay, Crystal Violet Assay, shRNA

Journal: Experimental and Therapeutic Medicine

Article Title: KDM6A suppresses hepatocellular carcinoma cell proliferation by negatively regulating the TGF-β/SMAD signaling pathway

doi: 10.3892/etm.2020.9000

Figure Lengend Snippet: KDM6A overexpression suppresses hepatocellular carcinoma cell tumorigenesis in vivo . (A) Representative images of the tumors generated by empty vector- and KDM6A overexpression vector-transfected Huh7 cells. Tumor (B) volume and (C) weight. (D) The effect of KDM6A overexpression on the protein expression levels of TGF-β, p-smad2, Ki67 and PCNA in tumor samples. * P<0.05, ** P<0.01 and *** P<0.001 vs. vector. KDM6A, lysine demethylase 6A; TGF-β, transforming growth factor-β; p, phosphorylated; PCNA, proliferating cell nuclear antigen; ns, not significant.

Article Snippet: Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-KDM6A (1:1,000; cat. no. orb333886; Biorbyt Ltd.), anti-transforming growth factor (TGF)-β (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-smad2 (1:1,000; cat. no. 18338; Cell Signaling Technology, Inc.), anti-Smad2 (1:1,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), anti-p-smad4 (1:1,000; cat. no. 10231-8-AP; ProteinTech Group, Inc.), anti-smad4 (1:1,000; cat. no. 10231-1-AP; ProteinTech Group, Inc.), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 10205-2-AP; ProteinTech Group, Inc.), anti-Ki67 (1:1,000; cat. no. 27309-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) and anti-Flag (1:4,000; cat. no. F7425; Sigma-Aldrich; Merck KGaA).

Techniques: Over Expression, In Vivo, Generated, Plasmid Preparation, Transfection, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: KDM6A suppresses hepatocellular carcinoma cell proliferation by negatively regulating the TGF-β/SMAD signaling pathway

doi: 10.3892/etm.2020.9000

Figure Lengend Snippet: KDM6A negatively regulates the TGF-β/SMAD signaling pathway. (A) The effect of KDM6A overexpression on the expression of TGF-β, p-smad2, p-smad4, Ki67 and PCNA in Huh7 and LM3 cells was (A) determined by western blotting and (B) semi-quantified. The effect of KDM6A knockdown on the expression of TGF-β, p-smad2, p-smad4, Ki67 and PCNA in YY-8103 and SNU-398 was (C) determined by western blotting and (D) semi-quantified. ** P<0.01 and *** P<0.001 vs. vector or SCR. KDM6A, lysine demethylase 6A; TGF-β, transforming growth factor-β; p, phosphorylated; PCNA, proliferating cell nuclear antigen; SCR, scrambled; sh, short hairpin RNA; ns, not significant.

Article Snippet: Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-KDM6A (1:1,000; cat. no. orb333886; Biorbyt Ltd.), anti-transforming growth factor (TGF)-β (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-smad2 (1:1,000; cat. no. 18338; Cell Signaling Technology, Inc.), anti-Smad2 (1:1,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), anti-p-smad4 (1:1,000; cat. no. 10231-8-AP; ProteinTech Group, Inc.), anti-smad4 (1:1,000; cat. no. 10231-1-AP; ProteinTech Group, Inc.), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 10205-2-AP; ProteinTech Group, Inc.), anti-Ki67 (1:1,000; cat. no. 27309-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) and anti-Flag (1:4,000; cat. no. F7425; Sigma-Aldrich; Merck KGaA).

Techniques: Over Expression, Expressing, Western Blot, Plasmid Preparation, shRNA