Journal: G3: Genes|Genomes|Genetics

Article Title: RNA sequencing-based screen for reactivation of silenced alleles of autosomal genes

doi: 10.1093/g3journal/jkab428

Figure Lengend Snippet: Hit validation for 5-aza-dC. (A and B) Confirmation of Screen-seq results for 5-aza-dC-treated cells using an orthogonal method to measure AI. cDNA samples from day 7 of screening were assessed using ddPCR with allele-specific fluorescent probes. (A) Scatterplots for 20,000 droplets targeting the readout gene, Col6a5 . 5-aza-dC concentration is shown in the plots. Black : empty droplets; blue : droplets with the Cast paternal allele amplified (labeled by FAM fluorophore); red : droplets with the 129 maternal allele amplified (labeled by HEX fluorophore). Ratio of red: blue droplets are shown. AI value written in red is the maternal AI. Note that the double-positive droplets (orange) contained both maternal and paternal templates; a small number of such double-positives is expected with higher concentrations of biallelic template. These droplets are ignored in the quantitative analysis. (B) left— summary of AI measurements shown in (A) for Col6a5 on day 7; right— summary of AI measurements for Dnajc12 on day 7. (C and D) Biological replicate of Abl.1 cells were treated with 5-aza-dC and AI was measured using ddPCR. (C) Scatterplots representation as shown in (A) after 2 days of exposure. (D) Summary of AI measurement for Col6a5 (left) and Dnajc12 (right) after 2, 5, and 7 days of exposure (denoted by color). Gray vertical dashed lines for Col6a5 dose-response were used to determine “low,” “medium,” and “high” 5-aza-dC concentrations for the future experiments. Results for readout gene Adnp2 are in . (E–G) Analysis of Dnmt1 knock-down (KD) in Abl.1 cells. (E) Real-time quantitative PCR (RT-qPCR) analysis of Dnmt1 relative expression (expression in the empty vector control, normalized to Nono , taken as 1.0). Abl.1 cells were transduced with an empty plKO vector (control) or with two separate Dnmt1 shRNA knockdown constructs ( Dnmt1 KD construct 1 or 2) and grown for 2 days. Transduced cells were then selected by growing in the presence of a selection antibiotic for an additional 17 days. RT-qPCR quantification was performed on cells collected 19 days after transduction. Mean and SEM for three technical replicates are shown. (F) Representative scatterplots show AI measurement for Col6a5 in the transduced Abl.1 cells. AI was measured using ddPCR. (G) Summary of the AI measurement for Col6a5 (left) and Dnajc12 (right) after Dnmt1 KD.

Article Snippet: Two shRNA vectors targeting Dnmt1 (SHR000038801.1_TRC001.1 and SHR000373188.1_TRC005.1) and a control empty vector (NUL003.3_TRC021.1) packaged in lentiviral vectors obtained from the Genetic Perturbation Platform at the Broad Institute were tested.

Techniques: Biomarker Discovery, Concentration Assay, Amplification, Labeling, Knockdown, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Plasmid Preparation, Control, Transduction, shRNA, Construct, Selection

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: BPH1 cells were transfected with 1μg/ml of DNMT expression vectors (OriGene) or constitutively active Akt (CA-Akt) #17-254 (Upstate Biotechnology, Lake Placid, NY) plasmids using Genjet vII reagent (Signagen, Gaithersburg, MD).

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: BPH1 cells were transfected with 1μg/ml of DNMT expression vectors (OriGene) or constitutively active Akt (CA-Akt) #17-254 (Upstate Biotechnology, Lake Placid, NY) plasmids using Genjet vII reagent (Signagen, Gaithersburg, MD).

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 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: BPH1 cells were transfected with 1μg/ml of DNMT expression vectors (OriGene) or constitutively active Akt (CA-Akt) #17-254 (Upstate Biotechnology, Lake Placid, NY) plasmids using Genjet vII reagent (Signagen, Gaithersburg, MD).

Techniques: Expressing, Activation Assay, Ubiquitin Proteomics, Methylation

Journal: PLOS One

Article Title: Glucocorticoid-Induced alterations in DNA methylation in the H19 promoter of Bone Marrow-Derived Mesenchymal Stem Cells are associated with the pathogenesis of osteonecrosis

doi: 10.1371/journal.pone.0345372

Figure Lengend Snippet: (A) When the concentration of 5-aza-dC reached 15 μM, cellular viability of hBMSCs from the ONFH group reached its peak at 24, 48, and 72 h (n = 6). (B) The cellular proliferative capacity was evaluated in hBMSCs of the control group, ONFH group, and 5-aza-dC treated group at 24, 48, and 72 h (n = 6). (C) qRT-PCR analysis revealed the relative expressions of H19 and DNMTs in undifferentiated hBMSCs of the control group, ONFH group, and 5-aza-dC treated group (n = 10). (D) Schematic diagram showed the location of 12 CpG sites within the analyzed region of the H19 promoter CpG island. (E) The BSP assay was used to analyze the methylation status of these CpG sites in the H19 promoter in hBMSCs of the three group.In the resulting methylation graph, each column represents a single individual subject sample, and each row corresponds to one of the 12 specific CpG sites analyzed, ordered by their genomic location. Empty dots: unmethylated CpGs; black dots: methylated CpGs. (F–I) qRT-PCR analysis showed the relative expressions of H19 (F) , Dnmt1 (G) , Dnmt3a (H) , and Dnmt3b (I) at days 3, 7, and 14 during osteogenic differentiation (n = 10). (J–M) qRT-PCR analysis showed the relative expressions of H19 (J) , Dnmt1 (K) , Dnmt3a (L) , and Dnmt3b (M) at days 3, 7, and 14 during adipogenic differentiation (n = 10).Statistical analysis was performed using one/two-way ANOVA with Bonferroni’s post-hoc test. Data are presented as mean ± SD, #P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: A short hairpin RNA (shRNA) targeting Dnmt1 was synthesized and cloned into a pLVX vector (Cyagen, Guangzhou, China) for the knockdown of Dnmt1 in rat BMSCs (rBMSCs).

Techniques: Concentration Assay, Control, Quantitative RT-PCR, PCR-BSP Assay, Methylation

Journal: PLOS One

Article Title: Glucocorticoid-Induced alterations in DNA methylation in the H19 promoter of Bone Marrow-Derived Mesenchymal Stem Cells are associated with the pathogenesis of osteonecrosis

doi: 10.1371/journal.pone.0345372

Figure Lengend Snippet: (A–F) Representative western blot bands and relative quantification of proteins indicated that Dnmt1 siRNA (A–B) , Dnmt3a siRNA (C–D) , and Dnmt3b siRNA (E–F) efficiently decreased the protein levels of Dnmt1 , Dnmt3a and Dnmt3b in hBMSCs of the ONFH group. β-actin was used as a loading control. (G) qRT-PCR analysis showed the relative expression of H19 in hBMSCs after DNMT knockdown. (H) BSP analysis was used to detect the methylation status of the H19 promoter. (I–J) Representative western blot images and quantification of relative protein expression indicated that overexpression of Dnmt1 by transfection efficiently increased Dnmt1 expression in hBMSCs. β-actin was used as a loading control. (K) qRT-PCR analysis showed the expression differences of H19 in hBMSCs from three different treatment groups. (L) BSP analysis was used to detect the methylation status of the H19 promoter in hBMSCs of the three groups. Statistical analysis was performed using one/two-way ANOVA with Bonferroni’s post-hoc test. Data are presented as mean ± SD, n = 3 per group, #P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control; si/siRNA: small interfering RNA; oe: overexpression.

Article Snippet: A short hairpin RNA (shRNA) targeting Dnmt1 was synthesized and cloned into a pLVX vector (Cyagen, Guangzhou, China) for the knockdown of Dnmt1 in rat BMSCs (rBMSCs).

Techniques: Western Blot, Quantitative Proteomics, Control, Quantitative RT-PCR, Expressing, Knockdown, Methylation, Over Expression, Transfection, Negative Control, Small Interfering RNA

Journal: PLOS One

Article Title: Glucocorticoid-Induced alterations in DNA methylation in the H19 promoter of Bone Marrow-Derived Mesenchymal Stem Cells are associated with the pathogenesis of osteonecrosis

doi: 10.1371/journal.pone.0345372

Figure Lengend Snippet: (A–B) ARS staining (A) and quantification (B) were performed to measure the calcium deposits in hBMSCs after 14 days of induction of osteogenic differentiation. Scale bars: 100 μm. (C–D) ORO staining (C) and quantification (D) were used to evaluate the intracellular lipid accumulation in hBMSCs after 21 days of induction of adipogenic differentiation. Scale bars: 50 μm. (E–G) After DNMT knockdown, the representative western blot band (E) and quantitative analysis showed the relative expressions of RUNX2 and COL1A1 (F) , as well as FABP4 and PPARγ (G) . β-actin was used as the loading control. (H–I) ARS staining (H) and quantification (I) were performed to measure the calcium deposits in hBMSCs. Scale bars: 100 μm. (J-K) ORO staining (J) and quantification (K) were used to evaluate the intracellular lipid accumulation in hBMSCs. Scale bars: 50 μm. (L–N) After overexpression of Dnmt1 , the representative western blot band (L) and quantitative analysis showed the relative expressions of RUNX2 and COL1A1 (M) as well as FABP4 and PPARγ (N) . β-actin was used as the loading control. Statistical analysis was performed using one/two-way ANOVA with Bonferroni’s post-hoc test. Data are presented as mean ± SD, n = 3 per group, #P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. NC, negative control; si/siRNA: small interfering RNA; oe: overexpression.

Article Snippet: A short hairpin RNA (shRNA) targeting Dnmt1 was synthesized and cloned into a pLVX vector (Cyagen, Guangzhou, China) for the knockdown of Dnmt1 in rat BMSCs (rBMSCs).

Techniques: Staining, Knockdown, Western Blot, Control, Over Expression, Negative Control, Small Interfering RNA

Journal: PLOS One

Article Title: Glucocorticoid-Induced alterations in DNA methylation in the H19 promoter of Bone Marrow-Derived Mesenchymal Stem Cells are associated with the pathogenesis of osteonecrosis

doi: 10.1371/journal.pone.0345372

Figure Lengend Snippet: (A) Schematic diagram of the experimental design for investigating the effects of implantation with Dnmt1 -knockdown or H19 -overexpression rBMSCs on the femoral heads of MPS-treated rats. (B) Fluorescence microscopy was used to observe infection efficiency in primary rBMSCs. Scale bars: 100 μm. (C) Western blot analysis showed that Dnmt1 shRNA efficiently decreased the protein expression levels of Dnmt1 in rBMSCs of MPS-treated rats. β-actin was used as a loading control. (D) Representative micro-CT images of the femoral head of each treatment group at week 6 after rBMSC transplantation are shown. (E–H) Micro-CT quantitative results are expressed as BV/TV (E) , Tb.Th (F) , Tb.N (G) , and Tb.Sp (H) (n = 6). (I) Representative images of H&E staining for each treatment group at week 6 after rBMSC implantation were shown and osteonecrosis was characterized by the empty lacunae (black arrow) or pyknotic nucleus of osteocytes (blue arrow) in trabecular bone. Scale bars: 25 μm. (J–K) Representative IHC images and quantitative analysis of positive stain of COL1A1 in the femoral head of each treatment group were shown (n = 6). Scale bars: 25 μm. (L–M) Representative IHC images and quantitative analysis of positive stain of FABP4 in the femoral head of each treatment group are shown (n = 6). Scale bars: 25 μm. Statistical analysis was performed using one/two-way ANOVA with Bonferroni’s post-hoc test. Data are presented as mean ± SD, #P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC: normal control, sh/shRNA: short hairpin RNA, sh-Ctrl: control shRNA.

Article Snippet: A short hairpin RNA (shRNA) targeting Dnmt1 was synthesized and cloned into a pLVX vector (Cyagen, Guangzhou, China) for the knockdown of Dnmt1 in rat BMSCs (rBMSCs).

Techniques: Knockdown, Over Expression, Fluorescence, Microscopy, Infection, Western Blot, shRNA, Expressing, Control, Micro-CT, Transplantation Assay, Staining

Journal: PLOS One

Article Title: Glucocorticoid-Induced alterations in DNA methylation in the H19 promoter of Bone Marrow-Derived Mesenchymal Stem Cells are associated with the pathogenesis of osteonecrosis

doi: 10.1371/journal.pone.0345372

Figure Lengend Snippet: A schematic diagram summarizing the proposed role of the Dnmt1/H19/GSK-3β axis in the pathogenesis of GC-induced ONFH.

Article Snippet: A short hairpin RNA (shRNA) targeting Dnmt1 was synthesized and cloned into a pLVX vector (Cyagen, Guangzhou, China) for the knockdown of Dnmt1 in rat BMSCs (rBMSCs).

Techniques:

Journal: BMC Cancer

Article Title: DNMT1 blocks SOX21-repressed CKS2 transcription to promote gastric cancer progression

doi: 10.1186/s12885-025-14577-z

Figure Lengend Snippet: High expression of DNMT1 promotes DNA methylation of the SOX21 promoter. A Prediction of CpG islands in the SOX21 promoter region using the MethPrimer database. B The methylation of the SOX21 promoter region was predicted on the Mexpress database. C The correlation between DNMT3A, DNMT1, DNMT3B, DNMT3L, and SOX21 methylation levels in GC from the Meth450 platform in the LinkOmics database using Spearman analysis. D DNMT1 expression was predicted in the STAD-UALCAN database. E The protein expression of DNMT1 in adjacent and tumor tissues in GC patients was examined using western blot analysis ( n = 13). F The protein expression of DNMT1 in GC cells and GES-1 cells was examined using western blot analysis. G The mRNA expression of DNMT1 in GC cells infected with shRNAs targeting DNMT1 was examined using RT-qPCR. H The protein expression of DNMT1 in GC cells infected with shRNAs targeting DNMT1 was examined using western blot analysis. I The methylation level of the SOX21 promoter in GC cells infected with sh-NC or sh-DNMT1 #2 was examined using the MSP assay. J The binding relation between DNMT1 and the SOX21 promoter was verified using a ChIP assay. K The binding relation between DNMT1 and the SOX21 promoter was examined using luciferase assays. L The mRNA expression of SOX21 in GC cells infected with sh-DNMT1 #2 was examined using RT-qPCR. M The protein expression of SOX21 in GC cells infected with sh-DNMT1 #2 was examined using western blot analysis. Paired or unpaired t-tests were used to compare the data between two groups, and ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Article Snippet: Lentiviral vectors containing shRNAs targeting DNMT1 or SOX21 (Table ), overexpression of SOX21, and overexpression of CKS2 (all from VectorBuilder, Guangzhou, Guangdong, China) were used to infect AGS and NCI-N87 cells, and Polybrene was added (1 μL/mL, C0351-1 ml, Beyotime, Shanghai, China).

Techniques: Expressing, DNA Methylation Assay, Methylation, Western Blot, Infection, Quantitative RT-PCR, MSP Assay, Binding Assay, Luciferase

Journal: BMC Cancer

Article Title: DNMT1 blocks SOX21-repressed CKS2 transcription to promote gastric cancer progression

doi: 10.1186/s12885-025-14577-z

Figure Lengend Snippet: Silencing of SOX21 abates the anti-proliferative and pro-apoptotic properties of sh-DNMT1 on GC cells. A The mRNA expression of SOX21 in GC cells infected with sh-DNMT1 + shRNAs targeting SOX21 was examined using RT-qPCR. B The protein expression of SOX21 in GC cells infected with sh-DNMT1 + shRNAs targeting SOX21 was examined using western blot analysis. C The EdU-positive GC cells after infection. D The OD value of GC cells was read at 0, 24, 48, and 72 h using the CCK-8 assay. E The migration and invasion of GC cells were examined using the Transwell assay. F Detection of apoptosis in GC cells by TUNEL assay. ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Article Snippet: Lentiviral vectors containing shRNAs targeting DNMT1 or SOX21 (Table ), overexpression of SOX21, and overexpression of CKS2 (all from VectorBuilder, Guangzhou, Guangdong, China) were used to infect AGS and NCI-N87 cells, and Polybrene was added (1 μL/mL, C0351-1 ml, Beyotime, Shanghai, China).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Migration, Transwell Assay, TUNEL Assay

Journal: BMC Cancer

Article Title: DNMT1 blocks SOX21-repressed CKS2 transcription to promote gastric cancer progression

doi: 10.1186/s12885-025-14577-z

Figure Lengend Snippet: DNMT1/SOX21/CKS2 axis is involved in GC progression in vivo. A The representative images of subcutaneous xenograft tumors in nude mice and the tumor growth curve within 25 d. B The weight of tumors formed by AGS cells with sh-NC, sh-DNMT1 #2 + sh-NC, sh-DNMT1 #2 + sh-SOX21 #3, oe-SOX21 + oe-NC, or oe-SOX21 + oe-CKS2 at day 25. C DNMT1, SOX21, and CKS2 expression in the tumor tissues was examined using western blot analysis. D The representative immunohistochemical images and positive cells of Ki67 and cleaved-caspase-3 in the tumor tissues formed by AGS cells. ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means ( n = 6)

Article Snippet: Lentiviral vectors containing shRNAs targeting DNMT1 or SOX21 (Table ), overexpression of SOX21, and overexpression of CKS2 (all from VectorBuilder, Guangzhou, Guangdong, China) were used to infect AGS and NCI-N87 cells, and Polybrene was added (1 μL/mL, C0351-1 ml, Beyotime, Shanghai, China).

Techniques: In Vivo, Expressing, Western Blot, Immunohistochemical staining

Journal: Advanced Science

Article Title: TP53BP2 Promotes Placental Autophagy and Preeclampsia via G9a and DNMT1 Cooperatively Modulating E2F1

doi: 10.1002/advs.202516408

Figure Lengend Snippet: TP53BP2 was upregulated in placental trophoblasts from PE pregnancies. (A) KEGG signaling pathway histogram. (B) Cluster heatmap showing the top 20 autophagy‐related DEGs associated with autophagy (|fold change|≥2.0, P ≤0.05) in the placentas of PE and non‐PE pregnancies. Red and blue strips indicate upregulated and downregulated genes, respectively. (C) Box plot illustrating the expression of the top 20 DEGs in placentas from PE and non‐PE pregnancies. The blue and red box plots represent PE and non‐PE pregnancies, respectively. (D) The expression of TP53BP2 in placentas was detected via western blotting (PC, n = 40; PE, n = 45). (E) Representative immunohistochemical staining of TP53BP2 . Scale bar = 200 µm. (F) Double immunofluorescence staining showing colocalization of TP53BP2 (red) and CK‐7 (trophoblast marker, green). The nuclei were stained with DAPI (blue). Scale bar = 50 µm. (G) Autophagosomes in HTR8/Svneo and JEG‐3 cells transfected with Ad‐ TP53BP 2 or sh‐ TP53BP2 under hypoxic conditions were observed using transmission electron microscopy (TEM). Scale bar = 1000 nm. (H) Representative images of GFP‐RFP‐LC3 staining in HTR8/Svneo and JEG‐3 cells transfected with sh‐ TP53BP2 under hypoxic conditions (n = 3). Scale bar = 20 µm. (I) The expression of LC3B‐II and p62 in HTR8/Svneo and JEG‐3 cells transfected with Ad‐ TP53BP2 or sh‐ TP53BP2 under hypoxic conditions was detected by western blotting (n = 3). Data are presented as mean ± SD. Student's t ‐test (unpaired, two‐tailed) was used to compare two independent groups. ** P <0.01, *** P <0.001.

Article Snippet: Recombinant adeno‐associated virus (AAV) serotype 9 vectors carrying TP53BP2 short hairpin RNA (shRNA; AAV‐ shTP53BP2 ), DNMT1 shRNA (AAV‐ DNMT1 ), and G9a shRNA (AAV‐ shG9a ), or recombinant AAV9 vectors carrying a negative control (AAV‐shNC) were manufactured by GeneChem Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Double Immunofluorescence Staining, Marker, Transfection, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: Advanced Science

Article Title: TP53BP2 Promotes Placental Autophagy and Preeclampsia via G9a and DNMT1 Cooperatively Modulating E2F1

doi: 10.1002/advs.202516408

Figure Lengend Snippet: TP53BP2 is a potential therapeutic target for trophoblast autophagy in PE. (A) On gestational day 14, SD rats underwent surgical reduction of uterine perfusion pressure (RUPP), followed by a single injection of 10 µL of AAV‐ shTP53BP2 (1.5E+11) into the placenta. (B) Expression of TP53BP2 in the placentas of preeclamptic rats was detected by western blotting and qRT‒PCR. (C) Immunofluorescence staining showing TP53BP2 expression in the placentas of preeclamptic rats. Scale bar = 50 µm. (D) Immunohistochemical staining was used to detect the expression of TP53BP2 in the placentas of preeclamptic rats. Scale bar = 200 µm. (E) Systolic and diastolic blood pressure were measured via a noninvasive tail‐cuff blood pressure measurement system in preeclamptic rats. (F) Total urine protein levels were measured using a protein assay in preeclamptic rats. (G) Gross appearance and birth weight of the fetus at embryonic day 18.5 (E18.5). (H) H&E staining analysis of placental pathological changes in preeclamptic rats. Scale bar = 500 µm. (I) The expression of LC3B ‐II and p62 in the placentas of preeclamptic rats was detected by western blotting. (J) Immunofluorescence staining was used to detect the expression of LC3B and p62 in the placentas of preeclamptic rats. Scale bar = 50 µm. (K) Immunohistochemical staining was used to detect the expression of LC3B and p62 in the placentas of preeclamptic rats. Scale bar = 200 µm. Data are presented as mean ± SD. Student's t ‐test (unpaired, two‐tailed) was used to compare two independent groups. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Recombinant adeno‐associated virus (AAV) serotype 9 vectors carrying TP53BP2 short hairpin RNA (shRNA; AAV‐ shTP53BP2 ), DNMT1 shRNA (AAV‐ DNMT1 ), and G9a shRNA (AAV‐ shG9a ), or recombinant AAV9 vectors carrying a negative control (AAV‐shNC) were manufactured by GeneChem Inc. (Shanghai, China).

Techniques: Injection, Expressing, Western Blot, Immunofluorescence, Staining, Immunohistochemical staining, Two Tailed Test

Journal: Advanced Science

Article Title: TP53BP2 Promotes Placental Autophagy and Preeclampsia via G9a and DNMT1 Cooperatively Modulating E2F1

doi: 10.1002/advs.202516408

Figure Lengend Snippet: TP53BP2 enhances trophoblast autophagy by regulating Beclin‐1 expression. (A) Volcano plot of 2081 DEGs in HTR8/SVneo cells transfected with sh‐TP53BP2. The red dots and green dots indicate upregulated and downregulated gene expression, respectively (|fold change|≥2.0, P ≤0.05), and the black dots indicate unchanged genes. (B) Heatmap of the top 10 downregulated autophagy‐related genes in the placentas of preeclamptic rats injected with AAV‐ shTP53BP2 . (C) qRT‒PCR validation of the top 10 downregulated genes. (D) Expression of Beclin ‐1, LC3B ‐II, p62 and TP53BP2 in HTR8/SVneo and JEG‐3 cells transfected with Ad‐TP53BP2 and/or sh‐Beclin‐1 under hypoxic conditions was detected by western blotting. (E) Co‐immunoprecipitation (Co‐IP) assay followed by immunoblotting showing the interactions of Bcl ‐2 with TP53BP2 and Beclin ‐1 in HTR8/SVneo and JEG‐3 cells under hypoxic conditions.Cell lysates were subjected to immunoprecipitation with an anti‐ Bcl ‐2 antibody. (F) Co‐IP assay followed by immunoblotting showing the interactions of Bcl ‐2 with TP53BP2 and Beclin‐1 in HTR8/SVneo and JEG‐3 cells transfected with sh‐ TP53BP2 under hypoxic conditions. Data are presented as mean ± SD. Student's t ‐test (unpaired, two‐tailed) was used to compare two independent groups, and a two‐way ANOVA test was performed for comparisons of multiple groups. * P <0.05, ** P <0.01, *** P <0.001; # P <0.05, ## P <0.01.

Article Snippet: Recombinant adeno‐associated virus (AAV) serotype 9 vectors carrying TP53BP2 short hairpin RNA (shRNA; AAV‐ shTP53BP2 ), DNMT1 shRNA (AAV‐ DNMT1 ), and G9a shRNA (AAV‐ shG9a ), or recombinant AAV9 vectors carrying a negative control (AAV‐shNC) were manufactured by GeneChem Inc. (Shanghai, China).

Techniques: Expressing, Transfection, Gene Expression, Injection, Biomarker Discovery, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Two Tailed Test

Journal: Advanced Science

Article Title: TP53BP2 Promotes Placental Autophagy and Preeclampsia via G9a and DNMT1 Cooperatively Modulating E2F1

doi: 10.1002/advs.202516408

Figure Lengend Snippet: Correlation between TP53BP2 expression and clinicopathological characteristics of patients with PE. (A, B) Pearson's correlation analysis between TP53BP2 expression and LC3B ‐II or p62 levels in placentas from PE and non‐PE pregnancies. (C–G) Pearson's correlation analysis between TP53BP2 expression and systolic blood pressure (C) , diastolic blood pressure (D) , BMI (E) , gestational age (F) , and neonatal birth weight (G) in PE pregnancies and non‐PE pregnancies. (H) The cutoff value, sensitivity, and specificity were established using receiver operating characteristic (ROC) curves to evaluate the diagnostic value of TP53BP2 in PE pregnancies. The data are presented as the means ± SDs. ** P <0.01.

Article Snippet: Recombinant adeno‐associated virus (AAV) serotype 9 vectors carrying TP53BP2 short hairpin RNA (shRNA; AAV‐ shTP53BP2 ), DNMT1 shRNA (AAV‐ DNMT1 ), and G9a shRNA (AAV‐ shG9a ), or recombinant AAV9 vectors carrying a negative control (AAV‐shNC) were manufactured by GeneChem Inc. (Shanghai, China).

Techniques: Expressing, Diagnostic Assay

Journal: Advanced Science

Article Title: TP53BP2 Promotes Placental Autophagy and Preeclampsia via G9a and DNMT1 Cooperatively Modulating E2F1

doi: 10.1002/advs.202516408

Figure Lengend Snippet: DNA methylation represses TP53BP2 transcription via DNMT1. (A) The promoter activity of TP53BP2 was evaluated using a dual‐luciferase reporter assay. Different fragments of the TP53BP2 promoter (‐35/+1, ‐599/+1, ‐1018/+1, ‐1530/+1, ‐599/+641, and ‐2000/+1) were transfected into HEK293T cells with a Renilla luciferase vector (internal control), and the results are presented as firefly luciferase activity normalized to Renilla luciferase activity. (B) Dual‐luciferase reporter assay analysis of the luciferase activities of the TP53BP2 promoter (‐599/‐35) in HTR8/SVneo and JEG‐3 cells subjected to hypoxic conditions. (C) Methylation of the TP53BP2 promoter in HEK293T cells. Following methylation with SssI , HhaI , or HpaII methylases, TP53BP2 promoter fragments were digested with McrBC (a methylation‐specific restriction enzyme), HpaII or HhaI (a methylation‐sensitive restriction enzyme) to confirm the methylation status of the TP53BP2 promoter construct. (D) The activity of the TP53BP2 proximal promoter methylated with SssI , HhaI , or HpaII methylases was assessed using a luciferase reporter assay in HEK293T cells transfected with luciferase reporter constructs. (E, F) The DNA methylation levels of the TP53BP2 promoter were evaluated using methylation‐specific PCR (MSP) in placentas and in HTR8/SVneo and JEG‐3 cells under hypoxic conditions. U: unmethylated; M: methylated. (G) The DNA methylation levels of the TP53BP2 promoter were evaluated via bisulfite sequencing PCR (BSP) in HTR8/SVneo cells under hypoxic conditions. White cycle, unmethylated CpG dinucleotides; black cycle, methylated CpG dinucleotides. The percentage of methylation at each CpG dinucleotide was calculated as the number of methylated clones at each CpG site divided by the total number of clones at the same CpG site and is shown in the right panel. (H) The DNA methylation level of the TP53BP2 promoter was evaluated via BSP in HTR8/SVneo cells treated with DC_05 (a DNMT1 ‐specific inhibitor), Theaflavin‐3, 3’‐digallate (TFD, a DNMT3a ‐specific inhibitor) or Nanomycin A (NA, a DNMT3b ‐specific inhibitor) under hypoxic conditions. (I) The DNA methylation level of the TP53BP2 promoter was evaluated via BSP in HTR8/SVneo cells transfected with sh‐ DNMT1 under hypoxic conditions. (J) Transcriptional activity of TP53BP2 in HTR8/SVneo cells transfected with sh‐ DNMT1 . (K) TP53BP2 expression was evaluated by western blotting in HTR8/SVneo cells transfected with sh‐ DNMT1 under hypoxic conditions. Data are presented as mean ± SD. Student's t ‐test (unpaired, two‐tailed) was used to compare two independent groups. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Recombinant adeno‐associated virus (AAV) serotype 9 vectors carrying TP53BP2 short hairpin RNA (shRNA; AAV‐ shTP53BP2 ), DNMT1 shRNA (AAV‐ DNMT1 ), and G9a shRNA (AAV‐ shG9a ), or recombinant AAV9 vectors carrying a negative control (AAV‐shNC) were manufactured by GeneChem Inc. (Shanghai, China).

Techniques: DNA Methylation Assay, Activity Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Control, Methylation, Construct, Methylation Sequencing, Clone Assay, Expressing, Western Blot, Two Tailed Test

Journal: Advanced Science

Article Title: TP53BP2 Promotes Placental Autophagy and Preeclampsia via G9a and DNMT1 Cooperatively Modulating E2F1

doi: 10.1002/advs.202516408

Figure Lengend Snippet: DNMT1 inhibits TP53BP2 expression by inversely modulating E2F1 in trophoblasts. (A) E2F1 expression in the placenta was detected using qRT‒PCR and western blotting. (B) Immunofluorescence staining was used to detect E2F1 expression (green) in placental trophoblasts. Scale bar = 50 µm. (C) TP53BP2 expression was detected by western blotting in HTR8/SVneo cells transfected with Ad‐ E2F1 or sh‐ E2F1 under hypoxic conditions. (D) Enrichment of E2F1 at the promoter region of TP53BP2 in HTR8/SVneo cells under hypoxic conditions was examined using a ChIP assay with an E2F1 antibody. (E) A schematic diagram of the predicted E2F1 ‐binding sites in the TP53BP2 promoter region from the JASPAR database ( http://www.genereg.net/ ). The blue box represents the ‐33/‐22 site. The green box represents the ‐99/‐88 site. The yellow box represents the ‐368/‐357 site. (F) A ChIP assay was conducted to assess the binding of E2F1 to specific sites (‐33/‐22, ‐99/‐88, and ‐368/‐357) on the TP53BP2 promoter in HTR8/SVneo cells subjected to hypoxic conditions. (G) The promoter activities of TP53BP2 with the wild‐type (WT) or mutant ‐33/‐22 site (Mut1), ‐99/‐88 site (Mut2) and ‐368/‐357 site (Mut3) of E2F1 were determined via a luciferase reporter assay in HEK293T cells. (H) Co‐IP assay followed by immunoblotting showing the interactions between DNMT1 and E2F1 in HTR8/SVneo cells under hypoxic conditions. (I) A ChIP assay was performed to demonstrate that E2F1 binds to the TP53BP2 promoter in HTR8/SVneo cells transfected with sh‐ DNMT1 . Data are presented as mean ± SD. Student's t ‐test (unpaired, two‐tailed) was used to compare two independent groups. ** P <0.01, *** P <0.001.

Article Snippet: Recombinant adeno‐associated virus (AAV) serotype 9 vectors carrying TP53BP2 short hairpin RNA (shRNA; AAV‐ shTP53BP2 ), DNMT1 shRNA (AAV‐ DNMT1 ), and G9a shRNA (AAV‐ shG9a ), or recombinant AAV9 vectors carrying a negative control (AAV‐shNC) were manufactured by GeneChem Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Transfection, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: Advanced Science

Article Title: TP53BP2 Promotes Placental Autophagy and Preeclampsia via G9a and DNMT1 Cooperatively Modulating E2F1

doi: 10.1002/advs.202516408

Figure Lengend Snippet: TP53BP2 is inhibited by G9a‐mediated histone methylation in trophoblasts. (A) H3K4me1 , H3K4me2 , H3K4me3 , H3K9me2 , H3K9me3 , H3K27me3 and H3K36me3 modifications were detected in the TP53BP2 promoter region. (B) ChIP assays were conducted to assess the enrichment of H3K4me1 , H3K4me2 , H3K4me3 , H3K9me2 , H3K9me3 , H3K27me3 and H3K36me3 at the TP53BP2 promoter in HTR8/SVneo cells under hypoxic conditions. (C) Immunofluorescence staining was used to detect the expression of H3K9me2 (green) in placental trophoblasts. Scale bar = 50 µm. (D, E) The mRNA expression levels of LSD1 , SUV39H1 , SUV39H2 and G9a were measured via qRT‒PCR in placentas and in HTR8/SVneo cells under hypoxic conditions. (F) Transcriptional activity of the TP53BP2 promoter in HTR8/SVneo cells transfected with sh‐G9a under hypoxic conditions. (G) Expression of TP53BP2 in HTR8/SVneo cells treated with BIX‐01294 (a G9a‐specific inhibitor) under hypoxic conditions was detected by western blotting. Data are presented as mean ± SD. Student's t ‐test (unpaired, two‐tailed) was used to compare two independent groups. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Recombinant adeno‐associated virus (AAV) serotype 9 vectors carrying TP53BP2 short hairpin RNA (shRNA; AAV‐ shTP53BP2 ), DNMT1 shRNA (AAV‐ DNMT1 ), and G9a shRNA (AAV‐ shG9a ), or recombinant AAV9 vectors carrying a negative control (AAV‐shNC) were manufactured by GeneChem Inc. (Shanghai, China).

Techniques: Methylation, Immunofluorescence, Staining, Expressing, Activity Assay, Transfection, Western Blot, Two Tailed Test

Journal: Advanced Science

Article Title: TP53BP2 Promotes Placental Autophagy and Preeclampsia via G9a and DNMT1 Cooperatively Modulating E2F1

doi: 10.1002/advs.202516408

Figure Lengend Snippet: DNMT1 and G9a cooperatively regulate TP53BP2 expression in PE. (A) Enrichment of E2F1 at the promoter region of TP53BP2 was analyzed via ChIP in HTR8/SVneo cells transfected with Ad‐ DNMT1 and/or Ad‐ G9a under hypoxic conditions. (B) BSP analysis was performed to determine the TP53BP2 DNA methylation level in HTR8/SVneo cells transfected with sh‐ DNMT1 and/or sh‐ G9a under hypoxic conditions. (C) H3K9me2 enrichment at the promoter region of TP53BP2 was analyzed via ChIP in HTR8/SVneo cells transfected with sh‐ DNMT1 and/or sh‐ G9a under hypoxic conditions. (D) Promoter transcription activity of TP53BP2 was analyzed using a dual‐luciferase reporter assay in HTR8/SVneo cells transfected with sh‐ DNMT1 and/or sh‐G9a under hypoxic conditions. (E) Western blotting was performed to determine TP53BP2 expression in HTR8/SVneo cells transfected with sh‐ DNMT1 and/or sh‐ G9a under hypoxic conditions. (F) Placental pathological changes in PE rats injected with AAV‐ shG9a and/or AAV‐ shDNMT1 were evaluated using H&E staining. Scale bars = 500 µm. (G) Noninvasive tail‐cuff blood pressure measurement system used to detect the systolic blood pressure and diastolic blood pressure of PE rats injected with AAV‐ shG9a and/or AAV‐ shDNMT1 . (H) Total urine protein levels in PE rats injected with AAV‐ shG9a and/or AAV‐ shDNMT1 . (I) Co‐IP assay followed by immunoblotting showing the interactions between G9a and DNMT1 or E2F1 in HTR8/SVneo cells under hypoxic conditions. (J) Immunofluorescence staining was used to measure the colocalization of DNMT1 (red) and E2F1 (green) or G9a (green) in HTR8/SVneo cells under hypoxic conditions. The nuclei were stained with DAPI. Scale bar = 20 µm. (K) Schematic diagram depicting the structure of DNMT1 and truncation mutants of the GST‐tagged DNMT1 fragments (GST‐Control, GST‐WT, GST‐1‐446, GST‐431‐703, GST‐643‐835, GST‐836‐1060, and GST‐1061‐1632). (L) The interactions between DNMT1 and G9a or between DNMT1 and E2F1 were examined via Co‐IP with an anti‐Myc antibody in HEK293T cells cotransfected with plasmids encoding different GST‐tagged DNMT1 fragments and plasmids encoding Myc‐tagged G9a (Myc‐ G9a ) or Flag‐tagged E2F1 (Flag‐ E2F1 ), respectively. (M) The transcriptional activity of the TP53BP2 promoter in HTR8/SVneo cells transfected with the Δ1‐446 mutation or the Δ1061‐1632 mutation. (N) The enrichment of E2F1 , DNMT1 , G9a and H3K9me2 at the promoter region of TP53BP2 in HTR8/SVneo cells transfected with wild‐type DNMT1 (WT), the G9a binding region deletion mutant DNMT1 (Δ1‐446) or the E2F1 binding region deletion mutant DNMT1 (Δ1061‐1632) was assessed via a ChIP assay under hypoxic conditions. Data are presented as mean±SD. Student's t ‐test (unpaired, two‐tailed) was used to compare two independent groups, and a two‐way ANOVA test was performed for comparisons of multiple groups. * P <0.05, ** P <0.01, *** P <0.001; # P <0.05.

Article Snippet: Recombinant adeno‐associated virus (AAV) serotype 9 vectors carrying TP53BP2 short hairpin RNA (shRNA; AAV‐ shTP53BP2 ), DNMT1 shRNA (AAV‐ DNMT1 ), and G9a shRNA (AAV‐ shG9a ), or recombinant AAV9 vectors carrying a negative control (AAV‐shNC) were manufactured by GeneChem Inc. (Shanghai, China).

Techniques: Expressing, Transfection, DNA Methylation Assay, Activity Assay, Luciferase, Reporter Assay, Western Blot, Injection, Staining, Co-Immunoprecipitation Assay, Immunofluorescence, Control, Mutagenesis, Binding Assay, Two Tailed Test

Journal: Advanced Science

Article Title: TP53BP2 Promotes Placental Autophagy and Preeclampsia via G9a and DNMT1 Cooperatively Modulating E2F1

doi: 10.1002/advs.202516408

Figure Lengend Snippet: TP53BP2 promotes placental autophagy and preeclampsia via G9a, and DNMT1 cooperatively modulates E2F1. The inhibition of TP53BP2 expression attenuates the progression of PE by inhibiting the autophagy of trophoblasts in SD rats; this is attributed to the fact that G9a‐mediated H3K9me2‐ and DNMT1 ‐mediated DNA hypomethylation suppressed the binding of E2F1 at the TP53BP2 promoter to suppress the expression of TP53BP2 transcription and subsequently inhibited the release of Beclin‐1 from the Bcl‐2‐Beclin‐1 complex.

Article Snippet: Recombinant adeno‐associated virus (AAV) serotype 9 vectors carrying TP53BP2 short hairpin RNA (shRNA; AAV‐ shTP53BP2 ), DNMT1 shRNA (AAV‐ DNMT1 ), and G9a shRNA (AAV‐ shG9a ), or recombinant AAV9 vectors carrying a negative control (AAV‐shNC) were manufactured by GeneChem Inc. (Shanghai, China).

Techniques: Inhibition, Expressing, Binding Assay

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: PC3 cells were transfected with DNMT shRNAs (OriGene Technologies Inc., Rockville, MD) and RASS F1A expression vector [34].

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: PC3 cells were transfected with DNMT shRNAs (OriGene Technologies Inc., Rockville, MD) and RASS F1A expression vector [34].

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: PC3 cells were transfected with DNMT shRNAs (OriGene Technologies Inc., Rockville, MD) and RASS F1A expression vector [34].

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 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: PC3 cells were transfected with DNMT shRNAs (OriGene Technologies Inc., Rockville, MD) and RASS F1A expression vector [34].

Techniques: Expressing, Activation Assay, Ubiquitin Proteomics, Methylation

Journal: Cancer & Metabolism

Article Title: Epigenetic repression of de novo cysteine synthetases induces intra-cellular accumulation of cysteine in hepatocarcinoma by up-regulating the cystine uptake transporter xCT

doi: 10.1186/s40170-024-00352-4

Figure Lengend Snippet: Epigenetic modifications of the 5’-flanking region of the cysteine synthetic genes in the hepatic tumors. A and B , The mRNA levels of the enzymes involved in cysteine synthesis ( A ) and DNA methyltransferases ( B ) in the liver and BNL 1ME A.7 R.1-formed tumors in mice. The expression levels were normalized to those of 18s . The values in the liver were set at 1.0. Each value represents the mean with S.D. ( n = 6). ** P < 0.01; significant difference between the two groups ( t 10 = − 15.397, P < 0.001 for Cbs ; t 10 = − 7.424, P < 0.001 for Cth ; t 10 = − 3.258, P = 0.009 for Dnmt1 ; t 10 = 5.041, P = 0.001 for Dnmt3a ; t 10 = − 3.438, P = 0.006 for Dnmt3b ; unpaired t -test, two sided). C and D , Methylation status of the 5’-flanking region in mice Cbs (C) and Cth (D) genes in the BNL 1ME A.7 R.1-formed tumors. Left panels show representative electropherograms of direct-bisulfite sequencing. Triangles indicate the methylation sites. Right panels show the quantification of methylation levels. Each value represents the mean with S.D. ( n = 4). ** P < 0.01, * P < 0.05; significant difference between the two groups (unpaired t -test, two sided)

Article Snippet: Small hairpin RNA (shRNA) expressing vectors against the mouse Slc7a11 , Dnmt1 , Dnmt3a , or Dnmt3b gene were purchased from VectorBuilder (Chicago, IL).

Techniques: Expressing, Methylation, Methylation Sequencing

Journal: Cancer & Metabolism

Article Title: Epigenetic repression of de novo cysteine synthetases induces intra-cellular accumulation of cysteine in hepatocarcinoma by up-regulating the cystine uptake transporter xCT

doi: 10.1186/s40170-024-00352-4

Figure Lengend Snippet: DNA methyltransferase suppresses the expression of cysteine synthetic enzymes in BNL 1ME A.7 R.1 cells. A and B , Induction of CBS and CTH expression by pharmacological inhibition of DNA methyltransferase activity. BNL 1ME A.7 R.1 cells were treated with 500 nM decitabine for 24 h. The mRNA and protein levels were normalized to those of 18s and β-ACTIN, respectively. The values in vehicle-treated cells were set at 1.0. Each value represents the mean with S.D. ( n = 4). * P < 0.05; significant difference between the two groups ( t 6 = 2.719, P = 0.035 for Cth mRNA; t 6 = 3.498, P = 0.013 for CBS protein; t 6 = 3.270, P = 0.017 for CTH protein; unpaired t -test, two sided). C and D , Induction of CBS and CTH expressions by down-regulation of DNA methyltransferase. BNL 1ME A.7 R.1 cells were transduced with lentivirus expressing shRNA against Dnmt1 , Dnmt3a , or Dnmt3b . The mRNA and protein levels in mock-transduced and Dnmt -knockdown (KD) cells were normalized to those of 18s and β-ACTIN, respectively. The values in mock-transduced cells were set at 1.0. Each value represents the mean with S.D. ( n = 3). ** P < 0.01, * P < 0.05; significant difference between the indicated groups ( F 3,8 = 160.673, P < 0.001 for Cbs mRNA; F 3,8 = 13.969, P = 0.002 for Cth mRNA; F 3,8 = 13.491, P = 0.002 for CBS protein; F 3,8 = 24.930, P < 0.001 for CTH protein; ANOVA with Tukey–Kramer’s post hoc test)

Article Snippet: Small hairpin RNA (shRNA) expressing vectors against the mouse Slc7a11 , Dnmt1 , Dnmt3a , or Dnmt3b gene were purchased from VectorBuilder (Chicago, IL).

Techniques: Expressing, Inhibition, Activity Assay, Transduction, shRNA, Knockdown

Journal: PLOS Pathogens

Article Title: NS1-mediated DNMT1 degradation regulates human bocavirus 1 replication and RNA processing

doi: 10.1371/journal.ppat.1012682

Figure Lengend Snippet: DNMT1 was knocked down by two shRNAs in HEK293T cells. Stably knockdown cells obtained using puromycin selection were transfected with pHBoV1-WH. (A) The NS1 or NP1 expression was quantified through western blotting, and GAPDH was used as loading control. Knockdown efficiencies were verified through western blotting. (B) HBoV replications were measured by Southern blotting as described above. (C) The reduced viral replication was further verified in DNMT1 knockdown cells through qRT-PCR quantification of Hirt DNA. *, p < 0.05. (D–E) The subcellular location of NS1 (D) and NP1 (E) in DNMT1-knockdown cells were detected by immunofluorescence as described in . Scale bars, 5 μm. (F–I) DNMT1 knockdown enhanced HBoV RNA processing. HBoV transcription in DNMT1-knockdown cells was evaluated through northern blotting (F). RPA analysis of HBoV RNAs spliced at D1 (G) or D3 (H) sites or polyadenylated at (pA)p (I) in DNMT1-kncokdown samples.

Article Snippet: The shRNAs specific for DNMT1 (1#, 5’-CGACTACATCAAAGGCAGCAA-3’, 2#, 5’-GCCCAATGAGACTGACATCAA-3’) were cloned into pLKO.1-TRC clone vector (Addgene plasmid 10878) and the lentiviruses were packaged by co-transfection with psPAX2 and pMD2.G into HEK293T cells.

Techniques: Stable Transfection, Knockdown, Selection, Transfection, Expressing, Western Blot, Control, Southern Blot, Quantitative RT-PCR, Immunofluorescence, Northern Blot