anti dnmt3b  (Cell Signaling Technology Inc)

Journal: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: SGI1027 and MS1129 are DNMT protein degraders independent of VHL and HIF (A) Immunoblot analysis of DNMT1/3A/3B/3L proteins in ccRCC cell lines ( n = 2 biological replicates). (B) mRNA analysis of DNMT1 , DNMT3A , DNMT3B , and DNMT3L in RCC10 cells treated with vehicle or SGI1027 for 2 days ( n = 2 biological replicates). (C–F) Immunoblot analyses of DNMT1/3A/3B proteins in RCC10 (C, n = 3 biological replicates), 786-O (D, n = 3 biological replicates), RCC4 (E, n = 2 biological replicates), and UM-RC-2 (F, n = 2 biological replicates) cells treated with vehicle or SGI1027 for 2 days. (G) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with vehicle or SGI1027 for indicated time ( n = 2 biological replicates). (H and I) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with vehicle or SGI1027 for 36 h, followed by co-treatment with vehicle, MG132 (6 h, n = 2 biological replicates, H), or MG262 (2 h, n = 2 biological replicates, I). (J and K) Immunoblot analysis of DNMT1/3A/3B proteins in isogenic RCC10 (J)/786-O (K) cells treated with vehicle or SGI1027 for 2 days ( n = 2 biological replicates). (L) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with vehicle or MS1129 for indicated time ( n = 2 biological replicates). (M and N) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with vehicle or MS1129 for 36 h, followed by co-treatment with vehicle, MG132 (6 h, n = 2 biological replicates, M), or MG262 (2 h, n = 2 biological replicates, N). (O) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with vehicle, MS1129, or MS1143 for 2 days ( n = 2 biological replicates). (P) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with CHX and/or SGI1027 for indicated time ( n = 2 biological replicates). (Q) Immunoblot analysis of DNMT1/3A/3B proteins in parental and DNMT1/3A/3B-deficient cells ( n = 2 biological replicates). (R) Representative images of death of parental and DNMT1/3A/3B-deficient cells. Scale bars, 100 μm. (S) Quantification of PI-positive cells in (R) ( n = 3 biological replicates, mean ± SEM). p value was determined by unpaired 2-tailed Student’s t test. See also .

Article Snippet: The supernatant was diluted in ChIP immunoprecipitation buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) and then subjected to immunoprecipitation overnight in the presence of Dynabeads (ThermoFisher) with anti-HIF-1α antibody (3 μg, home-made), anti-HIF-2α antibody (3 μg, home-made), anti-DNMT1 antibody (3 μg, 24206-1-AP, Proteintech), or anti-DNMT3A antibody (3 μg, 20954-1-AP, Proteintech), anti-DNMT3B antibody (3 μg, 26971-1-AP, Proteintech), or normal rabbit IgG (3 μg, 2729S, Cell Signaling Technology) at 4°C.

Techniques: Western Blot

Journal: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: dDNMT activates TRAIL-death receptor signaling in VHL -deficient ccRCC cells (A) Volcano plot of SGI1027-induced and -repressed genes in RCC10 cells ( n = 2 biological replicates). FC, fold change. (B) Biocarta pathway enrichment analysis of SGI1027-induced genes in RCC10 cells. (C) RT-qPCR analysis of TNFSF10 , TNFRSF10A , TNFRSF10B , and TNFRSF10D mRNA levels in RCC10 cells treated with vehicle or SGI1027 for 2 days ( n = 3 biological replicates). (D and E) Immunoblot analysis of TRAIL, DR4, DR5, DcR2, pro-caspase-10, and cleaved caspase-10 (C-caspase-10) proteins in isogenic RCC10 cells treated with vehicle, SGI1027 (D and E, n = 2 biological replicates), MS1129 (E, n = 2 biological replicates), or decitabine (E, n = 2 biological replicates) for 2 or 7 days. (F) Global m5C levels in RCC10 cells treated with vehicle or SGI1027 for 2 days by ELISA assay ( n = 3 biological replicates). (G–J) MeDIP-qPCR assay in RCC10 cells treated with vehicle or SGI1027 for 2 days ( n = 3 biological replicates). (K–M) DNMT1, DNMT3A, and DNMT3B ChIP-qPCR assay in RCC10 cells ( n = 3 biological replicates). (N) Scheme of dDNMT-activated apoptotic pathway. Data represent mean ± SEM. p value was determined by bioinformatics with edgeR (A) or gene set enrichment analysis (B), unpaired 2-tailed Student’s t test (C and F), two-way ANOVA with Tukey’s test (G–I), and one-way ANOVA with Dunnett’s test (K–M). See also and .

Article Snippet: The supernatant was diluted in ChIP immunoprecipitation buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) and then subjected to immunoprecipitation overnight in the presence of Dynabeads (ThermoFisher) with anti-HIF-1α antibody (3 μg, home-made), anti-HIF-2α antibody (3 μg, home-made), anti-DNMT1 antibody (3 μg, 24206-1-AP, Proteintech), or anti-DNMT3A antibody (3 μg, 20954-1-AP, Proteintech), anti-DNMT3B antibody (3 μg, 26971-1-AP, Proteintech), or normal rabbit IgG (3 μg, 2729S, Cell Signaling Technology) at 4°C.

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Methylated DNA Immunoprecipitation, ChIP-qPCR

Journal: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: dDNMT specifically kills patient-derived VHL -deficient ccRCC in mice (A) Tumor growth curves of VHL -deficient UTSW-PDX206, UTSW-PDX258, UTSW-PDX490, and UTSW-PDX26 in mice treated with vehicle (Veh) or SGI1027 for 10 days. (B) Tumor growth curves of VHL -WT UTSW-PDX416 and UTSW-PDX143 in mice treated with vehicle or SGI1027 for 10 days. (C) Kaplan-Meier survival curve of UTSW-PDX490-bearing mice ( n = 10 biological replicates). (D and E) Global m5C levels in UTSW-PDX206 (D) or UTSW-PDX258 (E) tumors harvested from mice after treatments by ELISA assay ( n = 5 biological replicates). (F) Immunoblot analysis of DNMT1, DNMT3A, DNMT3B, TRAIL, DR4, DR5, procaspase-10, C-caspase-3, and C-caspase-7 proteins in UTSW-PDX258 tumors harvested from mice after treatments ( n = 5 biological replicates). (G) Representative C-caspase-3 IHC in UTSW-PDX258 tumors. Scale bar, 100 μm. (H) Quantification of C-caspase-3-positive cells in (G) ( n = 5 biological replicates). (I) Immunoblot analysis of DNMT1, DNMT3A, DNMT3B, TRAIL, DR4, DR5, C-caspase-3, and VHL proteins in UTSW-PDX416 tumors harvested from mice after treatments ( n = 5 biological replicates). (J) Immunoblot analysis of DNMT1, DNMT3A, DNMT3B, and procaspase-10 proteins in UTSW-PDX206, UTSW-PDX258, and UTSW-PDX26 tumors ( n = 4–5 biological replicates). Data represent mean ± SEM. p value was determined by two-way ANOVA with Tukey’s test (A), log rank test (C), and unpaired 2-tailed Student’s t test (D, E, and H). See also and ; .

Article Snippet: The supernatant was diluted in ChIP immunoprecipitation buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) and then subjected to immunoprecipitation overnight in the presence of Dynabeads (ThermoFisher) with anti-HIF-1α antibody (3 μg, home-made), anti-HIF-2α antibody (3 μg, home-made), anti-DNMT1 antibody (3 μg, 24206-1-AP, Proteintech), or anti-DNMT3A antibody (3 μg, 20954-1-AP, Proteintech), anti-DNMT3B antibody (3 μg, 26971-1-AP, Proteintech), or normal rabbit IgG (3 μg, 2729S, Cell Signaling Technology) at 4°C.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot