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

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 1 Mahanine restores RASSF1A expression by demethylating its promoter and all three DNMTs control RASSF1A expression. A. PC3 cells were treated with DMSO control or 10 μM mahanine for 1 and 3 days. Methylation-specific PCR was performed to detect the methylated (M) and un-methylated (UM) status of RASSF1A promoter. B. PC3 cells were treated with DMSO control or 10 μM mahanine for 1 and 3 days, following which RASSF1A expression was assessed by RT-PCR. GAPDH was used as an internal control. C. PC3 cells were transfected with shRNA for DNMT1, DNMT3A, DNMT3B or scrambled shRNA. Forty-eight hours after transfection, cells were harvested for RT-PCR analyses to assess RASSF1A expression. GAPDH was used as an internal control. For DNMT3A, two shRNAs were used to confirm the result. D. BPH1 cells were transfected with expression vectors of DNMT1, DNMT3A, DNMT3B or empty vector control. Forty-eight hours after transfection cells were collected for RT-PCR analyses to determine RASSF1A, DNMT1, DNMT3A and DNMT3B expression levels. GAPDH was used as an internal control.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Expressing, Control, Methylation, Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA, Plasmid Preparation

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 2 Mahanine specifically down-regulates DNMT1 and DNMT3B. A. DNMT1, DNMT3A and DNMT3B cellular localization was visualized by immunofluorescent staining. PC3 cells were treated with DMSO (as control) or mahanine (10 μM) for 24 hours, following which they were fixed in methanol, incubated with the indicated antibodies, stained with Alexa Fluor 488-tagged secondary antibodies and counterstained with propidium iodide. Slides were then mounted and examined under a fluorescence microscope. The bright field images of PC3 cells treated with DMSO or mahanine (10 μM) for 24 hours are shown (right panel). B. Cytoplasmic and nuclear fractions were separated from PC3 cells treated with DMSO or 10 μM mahanine for 24 hours. The isolated fractions were subjected to Western blot analysis to assess DNMT expression. The fold change in the expression of the respective DNMTs as compared to the control is indicated at the bottom of each immunoblot. Nucleolin and β-actin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. C. PC3 and LNCaP cells were treated as indicated with DMSO or mahanine following which cells were lysed and the extracts were subjected to Western blot analysis to detect DNMT1, DNMT3B and DNMT3A protein levels (left). Quantitative estimations of the relative levels of DNMT1, DNMT3A and DNMT3B proteins were determined by densitometric measurements of immunoblots from three independent experiments after normalization with β-actin (right). Columns, mean; bars, SEM. *p < 0.05, significantly different from control. D. PC3 and LNCaP cells were treated with DMSO or 10 and 20 μM mahanine, respectively for 24 hours. Subsequently, cells were harvested for RT-PCR analysis to measure DNMT1 and DNMT3B expression. GAPDH was used as an internal control.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Staining, Control, Incubation, Fluorescence, Microscopy, Isolation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 3 Mahanine degrades DNMTs via the ubiquitin-proteasomal pathway. A. PC3 cells were treated with 10 μM mahanine and 20 μM Z-VAD-FMK for 24 hours after which cellular protein lysates were subjected to Western blot analysis to detect DNMT1 and DNMT3B protein levels, β-actin was used as a loading control. B. Chymotrypsin-like proteasomal activity was measured in PC3 cells treated as indicated with mahanine and MG132 for 24 hours. Columns, mean; bars, SEM. *p < 0.05, significantly different from DMSO control. C. LNCaP and PC3 cells were treated with the indicated doses of mahanine for 24 hours with or without MG132 (5 μM). Cell lysates were analyzed for DNMT1 and DNMT3B expression by Western blot. β-actin was used as a loading control. D. PC3 cells were treated with MG132 (5 μM) in the absence and presence of mahanine (10 μM) for 24 hours. Cell lysates were subjected to immunoprecipitation (IP) of DNMT1 or DNMT3B and immunoblotted (IB) for poly-ubiquitin, DNMT1 and DNMT3B.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Activity Assay, Expressing, Immunoprecipitation

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 4 Mahanine down-regulates pAkt levels and PI3K/Akt inhibitor wortmannin reduces DNMT1 and DNMT3B protein levels. A. PC3 and LNCaP cells were treated with mahanine as indicated. The cell lysates were subjected to Western blot analysis for phospho Akt (pAkt), total Akt. Β-actin was used as a loading control. B. PC3 and LNCaP cells were treated with wortmannin (1 μM) for 24 hours. Cell lysates were subjected to Western blot analysis to measure DNMT1, DNMT3B, pAkt and total Akt levels. β-actin was used as a loading control. The fold change in expression of the respective proteins compared to control is indicated below. C. PC3 cells were treated with DMSO or wortmannin (1 μM) for 24 hours. DNMT1 and DNMT3B cellular localization was visualized by immunofluorescent staining. After 24h of treatment, cells were fixed in methanol, incubated with the indicated antibodies, stained with Alexa Fluor 594-tagged secondary antibodies and counterstained with DAPI. Slides were then mounted and examined under a fluorescence microscope. The bright field images of PC3 cells treated with DMSO or wortmannin (1 μM) for 24 hours are shown (right panel).

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Western Blot, Control, Expressing, Staining, Incubation, Fluorescence, Microscopy

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 5 Mahanine disrupts the interaction of pAkt with DNMT1 and DNMT3B and constitutively active Akt (CA-Akt) stabilizes their cellular levels in the presence of mahanine. A. and B. LNCaP cells were treated with MG132 with or without 20 μM mahanine for 24 hours and the cell homogenates were subjected to co-immunoprecipitation (IP) for DNMT1 or DNMT3B and immunoblotted (IB) for phosphor-serine (pSer), pAkt, total Akt, DNMT1 and DNMT3B. C. BPH1 cells were transfected with an empty vector or a constitutively active Akt (CA-Akt) expression vector for 24 hours then were treated with or without mahanine (10 μM) for another 24 hours. Levels of total Akt, pAkt, DNMT1 and DNMT3B proteins were measured through Western blots. β-actin was used as a loading control. Quantitative estimations of relative levels of DNMT1 and DNMT3B proteins were determined by densitometric measurements of immunoblots from three independent experiments after normalization with β-actin. Columns, mean; bars, SEM. *p < 0.05, significantly different from DMSO treated control.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 6 Mahanine restores RASSF1A expression by degrading DNMTs via Akt. Prostate cancer cells express high levels of activated Akt, which phosphorylates and stabilizes DNMT1 and DNMT3B against proteasomal degradation. DNMTs enter the nucleus and methylate the promoter of RASSF1A gene to silence the expression of RASSF1A. Treatment of mahanine inhibits PDK1 and thereby prevents activation of Akt, which in turn compromises the stability of DNMTs, increases their ubiquitination and induces proteasomal degradation. In the absence of DNMT1 and DNMT3B, the RASSF1A promoter is demethylated and its expression is restored in prostate cancer cells. GF: Growth factor; RTK: Receptor tyrosine kinase; TFs: Transcription factors; P: Phosphorylated; M: Methylated; Ub: Ubiquitinated.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Expressing, Activation Assay, Ubiquitin Proteomics, Methylation

Journal: iScience

Article Title: MyoD is essential in rhabdomyosarcoma by promoting survival through differentiation and CYLD

doi: 10.1016/j.isci.2025.113149

Figure Lengend Snippet: MyoD suppresses death genes through DNA methyltransferases (A) Volcano plot from transcriptomic analysis representing downregulated (blue, n = 1358) and upregulated genes (red, n = 1116) from RH30-SR MyoD Δ cells compared to RH30-SR cells (fold change >25%, FDR <0.05). (B) GO analysis of upregulated genes in RH30-SR MyoD Δ cells compared to RH30-SR cells. (C) Volcano plot from ATAC-seq analysis representing differentially accessible chromatin—closed (blue) and open (red)—in RH30-SR MyoD Δ cells compared to RH30-SR cells (fold change >25%, FDR <0.05). (D) Cell death was measured following the treatment of RH30-SR cells with decitabine (1 μM) and TNF (5 ng/mL) compared to RH30-SR control cells treated with DMSO and PBS. Significance was determined through two-way ANOVA analysis, with Sidak’s multiple comparisons, n = 3. (E) Expression levels of DNMT1, DNMT3A, and DNMT3B in RH30 MyoD Δ and RH30 Myf5 Δ compared to RH30-Vector control cells analyzed by one-way ANOVA with Dunnett’s multiple comparisons, n = 3. (F) Western blots of MyoD, DNMT1, DNMT3A, and DNMT3B in RH30-SR Vector or MyoD Δ cells, using α−tubulin as a loading control. Arrowheads point to respective DNMTs. (G) MyoD ChIP-seq data identifying enrichment peaks on the DNMT3A gene from FN (RD) and FP (RH4) RMS cells, graphed above representative ATAC-seq data revealing loss of chromatin accessibility signal in highlighted regions of interest in RH30 MyoD Δ (purple) compared to RH30 Vector cells (blue) with accompanying composite plot of ATAC-seq signals of both conditions, n = 3. Signal is represented as reads per million mapped reads (RPM). (H) Visualization of MyoD ChIP-seq data with enrichment peaks identified as S1-S3 on DNMT1, DNMT3A and DNMT3B genes in FN (RD) and FP (RH4) RMS cells. (I) MyoD ChIP analysis performed, as percent of input, on regions S1–S3 in DNMT1, DNMT3A and DNMT3B genes, n = 2. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Article Snippet: Antibodies used in this study included, IκBα (Cell Signaling Technology Cat# 9242, RRID: AB_331623 ), α-tubulin (Thermo Fisher Scientific Cat# A11126, RRID: AB_2534135 ), p65/RelA (Santa Cruz Biotechnology Cat# sc-109, RRID: AB_632039 ), MyoD (Santa Cruz Biotechnology Cat# sc-377460, RRID: AB_2813894 ), Myf5 (Santa Cruz Biotechnology Cat# sc-518039), myogenin (Santa Cruz Biotechnology Cat# sc-52903, RRID: AB_784707 ), MRF4 (Santa Cruz Biotechnology sc-514379), MEF2C (Abcam Cat# ab211493, RRID: AB_2864417 ), MEF2D (Santa Cruz Biotechnology Cat# sc-271153, RRID: AB_10614669 ), MYHC (Sigma-Aldrich Cat# M8421, RRID: AB_477248 ), TnnT (Sigma-Aldrich Cat# SAB4200717, RRID: AB_2892079 ), CYLD (Cell Signaling Technology Cat# 4495, RRID: AB_10557111 ), p -RIPK1 (Proteintech Cat# 66854-1-Ig, RRID: AB_2882194 ), RIPK1 (Thermo Fisher Scientific Cat# PA5-20811, RRID: AB_11154790 ), DNMT1 (Novus Cat# NB100-56519, RRID: AB_838131 ), DNMT3A (Santa Cruz Biotechnology Cat# sc-365769, RRID: AB_10844010 ), DNMT3B (Cell Signaling Technology Cat# 67259, RRID: AB_2799723 ), and HRP conjugated secondary antibodies anti-mouse IgG (Promega Cat# W4021, RRID: AB_430834 ) and anti-rabbit IgG (Promega Cat# W4011, RRID: AB_430833 ).

Techniques: Control, Expressing, Plasmid Preparation, Western Blot, ChIP-sequencing

Journal: PLoS ONE

Article Title: 5-Aza-2’-deoxycytidine in the medial prefrontal cortex regulates alcohol-related behavior and Ntf3-TrkC expression in rats

doi: 10.1371/journal.pone.0179469

Figure Lengend Snippet: (A) Representative immunoblots and total protein image. Effects of alcohol exposure on the mRNA (B) and protein (C) expressions of DNMT1, DNMT3A and DNMT3B in the mPFC (n = 12/group). The data represent the mean ± SEM. *** p < 0.001, **** p < 0.0001 vs. water-exposure controls.

Article Snippet: The membranes were blocked with 5%(w/w) non-fat dried milk in Tris-buffered saline (TBS) (500 mM NaCl, 20 mM Tris-HCl pH 7.4) containing 0.05% Tween-20 for 1.5 h and incubated overnight with one of the following antibodies at 4°C: anti-DNMT1 polyclonal antibody (1:1000; Epigentek), anti-DNMT3A polyclonal antibody (1:400; Epigentek), anti-DNMT3B polyclonal antibody (1:500; Epigentek), anti-Ntf3 polyclonal antibody (1:1000; Abcam), anti-TrkC monoclonal antibody (1:1000, Cell Signaling Technology).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: 5-Aza-2’-deoxycytidine in the medial prefrontal cortex regulates alcohol-related behavior and Ntf3-TrkC expression in rats

doi: 10.1371/journal.pone.0179469

Figure Lengend Snippet: Fig 5A shows representative immunoblots and total protein image. 5-Aza-dc decreased the protein level of DNMT1 (B), whereas it reversed the alcohol-induced overexpression of DNMT3A (C) and DNMT3B (D). 5-Aza-dc prevented the Ntf3 reduction induced by chronic alcohol exposure (E) but had no effect on TrkC (F). (G) Differential expression of mRNA for DNMTs, Ntf3 and TrkC. * p < 0.05, ** p < 0.01, *** p < 0.001, differences compared to the Water+DMSO group; # p < 0.05, ## p < 0.01, ### p < 0.01, differences compared to the other corresponding groups; n = 8/group.

Article Snippet: The membranes were blocked with 5%(w/w) non-fat dried milk in Tris-buffered saline (TBS) (500 mM NaCl, 20 mM Tris-HCl pH 7.4) containing 0.05% Tween-20 for 1.5 h and incubated overnight with one of the following antibodies at 4°C: anti-DNMT1 polyclonal antibody (1:1000; Epigentek), anti-DNMT3A polyclonal antibody (1:400; Epigentek), anti-DNMT3B polyclonal antibody (1:500; Epigentek), anti-Ntf3 polyclonal antibody (1:1000; Abcam), anti-TrkC monoclonal antibody (1:1000, Cell Signaling Technology).

Techniques: Western Blot, Over Expression, Quantitative Proteomics

Journal: ACS nano

Article Title: Single-Cell Digital Lysates Generated by Phase-Switch Microfluidic Device Reveal Transcriptome Perturbation of Cell Cycle

doi: 10.1021/acsnano.8b01272

Figure Lengend Snippet: Perturbation of the cell cycle by DNMT3B knockdown. DNMT3B was suppressed with siRNA and the numbers of cells in different cell cycle stages were compared to cells treated with control siRNA. The distribution of cells in each cell cycle phase was altered by siRNA silencing of DNMT3B at 6, 24, and 48 h post-siRNA suppression. There were significantly more cells in the transition stages of S phase (yellow) at all time points, and there were fewer cells in G1 phase (red) at 6 and 24 h. There were fewer cells in M phase (colorless) at 6 and 48 h post-knockdown. Solid columns: control siRNA; Striped columns: DNMT3B knock-down. Two representative fluorescent images are presented (bottom panel) to show alternation of cells at different cell cycle stages at 24 h.

Article Snippet: The siRNAs to DNMT3B (Santa Cruz) were transfected into the cells according to the manufacturer’s instructions with slight modification.

Techniques: Knockdown, Control