Journal: Journal of Biochemistry

Article Title: DAXX promotes SUMOylation of chromatin-trapped DNMT1

doi: 10.1093/jb/mvag011

Figure Lengend Snippet: Xenopus egg extract recapitulates 5-aza-dCTP-induced SUMOylated DNMT1 accumulation on chromatin. (A) Xenopus sperm nuclei were incubated in egg extracts supplemented with or without 50 μM 5-aza-dCTP for indicated periods. Isolated chromatins were analyzed by immunoblotting with the indicated antibodies. (B) Immunoblotting results using indicated antibodies following DNMT1 immunoprecipitation of chromatin treated or untreated with 5-aza-dCTP for 60 min. The input results were shown in the lower panels.

Article Snippet: For DNMT1 inhibition, egg extracts were incubated with 50 μM 5-aza-dCTP (Jena Bioscience) or with 100 μM GSK-3484862 (MedChemExpress).

Techniques: Incubation, Isolation, Western Blot, Immunoprecipitation

Journal: Journal of Biochemistry

Article Title: DAXX promotes SUMOylation of chromatin-trapped DNMT1

doi: 10.1093/jb/mvag011

Figure Lengend Snippet: DAXX accumulates on DNMT1-trapped chromatin in a SUMO-dependent manner identified by CHROMASS. (A) Volcano plot showing chromatin mass spectrometry analysis of protein recruitment to chromatins treated with or without 5-aza-dCTP ( n = 3 technical replicates). Proteins highlighted in blue were identified in the comparison between 5-aza-dCTP+UBC9-DN and control groups and represent factors whose accumulation on chromatin is associated with SUMOylation. (B) Scatter plot showing protein abundance ratios derived from indicated comparisons in the axis titles.

Article Snippet: For DNMT1 inhibition, egg extracts were incubated with 50 μM 5-aza-dCTP (Jena Bioscience) or with 100 μM GSK-3484862 (MedChemExpress).

Techniques: Mass Spectrometry, Comparison, Control, Quantitative Proteomics, Derivative Assay

Journal: Journal of Biochemistry

Article Title: DAXX promotes SUMOylation of chromatin-trapped DNMT1

doi: 10.1093/jb/mvag011

Figure Lengend Snippet: DAXX interacts with SUMOylated DNMT1 on chromatin through SIMs. (A) The multiple sequence alignment of DAXX from different indicated species. DAXX contains SUMO-interacting motifs (SIMs), four-helix bundle (4HB), histone binding domain (HBD) and acidic residue-rich domain ( ad ). (B) Xenopus sperm nuclei were incubated in egg extracts supplemented with buffer, 5-aza-dCTP or 5-aza-dCTP and SUMO inhibitor ML-792. Isolated chromatin was analyzed by immunoblotting with indicated antibodies. (C) Xenopus sperm nuclei were incubated in mock-depleted or DNMT1-depleted extracts in the presence of buffer or 5-aza-dCTP. Isolated chromatin was analyzed by immunoblotting with indicated antibodies. (D) 3× FLAG-tagged xDAXX WT, 3× FLAG-tagged xDAXX SIM1 mutants (I7AI8A point mutant or 1–14 deletion mutant) or 3× FLAG-tagged xDAXX SIM2 mutants (V911A/I912A point mutant or 909–916 deletion mutant) were added to DAXX-depleted extracts. (E) Xenopus sperm nuclei were incubated in mock-depleted or DAXX-depleted extracts supplemented with xDAXX WT, xDAXX SIM1 mutants or xDAXX SIM2 mutants in the presence of buffer or 5-aza-dCTP.

Article Snippet: For DNMT1 inhibition, egg extracts were incubated with 50 μM 5-aza-dCTP (Jena Bioscience) or with 100 μM GSK-3484862 (MedChemExpress).

Techniques: Sequencing, Binding Assay, Residue, Incubation, Isolation, Western Blot, Mutagenesis

Journal: Journal of Biochemistry

Article Title: DAXX promotes SUMOylation of chromatin-trapped DNMT1

doi: 10.1093/jb/mvag011

Figure Lengend Snippet: DAXX promotes SUMOylation of chromatin-trapped DNMT1. (A) Immunoblotting results of egg extracts using indicated antibodies. 3× FLAG-tagged xDAXX WT was added to xDAXX-depleted egg extracts. (B) Chromatin fractions were isolated from mock-depleted or xDAXX-depleted egg extracts treated or untreated with 5-aza-dCTP for the indicated times, with or without adding 3× FLAG-tagged xDAXX. DNMT1 SUMOylation was assessed by immunoblotting using anti-DNMT1 antibodies.

Article Snippet: For DNMT1 inhibition, egg extracts were incubated with 50 μM 5-aza-dCTP (Jena Bioscience) or with 100 μM GSK-3484862 (MedChemExpress).

Techniques: Western Blot, Isolation

Journal: The FASEB Journal

Article Title: Aberrant DNMTs Promote TXNIP Upregulation and Ovarian Fibrosis in PCOS

doi: 10.1096/fj.202505030R

Figure Lengend Snippet: 5‐Aza treatment inhibits TXNIP and ovarian fibrosis in PCOS‐like rats. Rats received DHEA in the presence or absence of 5‐Aza treatment ( n = 8 in each group). (A) Ovarian and follicular morphology was assessed using H&E staining. (B) TXNIP expression in ovaries was analyzed using immunohistochemical staining. (C) Protein levels of DNMTs and TXNIP in two representative ovaries of each group were assessed using western blot (left panel). Band intensities were quantified ( n = 3; right panel). (D) Protein levels of pro‐fibrotic factors (collagen I, β ‐catenin, P‐SMAD3, α ‐SMA, and TGF‐ β ) in two representative ovaries of each group were assessed using western blot (left panel). Band intensities were quantified ( n = 3; right panel). Data are shown as the mean ± SD. * p ≤ 0.05, ** p ≤ 0.01. Each treatment group was compared with the control group. DHEA, dehydroepiandrosterone; DNMT1, DNA methyltransferase 1; DNMT3A, DNA methyltransferase 3A; DNMT3B, DNA methyltransferase 3B; TXNIP, thioredoxin‐interacting protein.

Article Snippet: To establish an in vitro hyperandrogenic PCOS‐like model, cells were treated with various concentrations (0.5, 2, and 5 μM) of dihydrotestosterone (DHT; Meilun Biological Technology Corporation, China) for 48 h. For determining the regulation of TXNIP, 5‐Aza (10 μM; MCE) or flutamide (20, 50 μM; Selleck, China) was added 24 h after the initiation of DHT treatment.

Techniques: Staining, Expressing, Immunohistochemical staining, Western Blot, Control

Journal: The FASEB Journal

Article Title: Aberrant DNMTs Promote TXNIP Upregulation and Ovarian Fibrosis in PCOS

doi: 10.1096/fj.202505030R

Figure Lengend Snippet: 5‐Aza treatment inhibits hyperandrogenism‐induced upregulation of TXNIP and pro‐fibrotic factors in GCs. Primary GCs were treated with DHT followed by 5‐Aza and MG132 ( n = 3 in each group). (A) Protein levels of DNMTs and TXNIP in GCs were assessed using western blot (left panel). Band intensities were quantified ( n = 3; right panel). (B‐D) Levels of DNMT1, DNMT3A, and TXNIP in GCs were analyzed using immunofluorescence staining (60×) (left panel). Fluorescence intensities were quantified ( n = 3; right panel). (E) Protein levels of pro‐fibrotic factors (collagen I, β ‐catenin, P‐SMAD3, α ‐SMA, and TGF‐ β ) in GCs were assessed using western blot. (F) Band intensities were quantified (E) ( n = 3). (G) Protein levels of TXNIP in GCs were assessed using western blot (upper panel). Band intensities were quantified ( n = 3; lower panel). Data are shown as the mean ± SD. * p ≤ 0.05, ** p ≤ 0.01. Each treatment group was compared with the control group. DHT, dihydrotestosterone; DNMT1, DNA methyltransferase 1; DNMT3A, DNA methyltransferase 3A; DNMT3B, DNA methyltransferase 3B; N.S, not significant; TXNIP, thioredoxin‐interacting protein.

Article Snippet: To establish an in vitro hyperandrogenic PCOS‐like model, cells were treated with various concentrations (0.5, 2, and 5 μM) of dihydrotestosterone (DHT; Meilun Biological Technology Corporation, China) for 48 h. For determining the regulation of TXNIP, 5‐Aza (10 μM; MCE) or flutamide (20, 50 μM; Selleck, China) was added 24 h after the initiation of DHT treatment.

Techniques: Western Blot, Immunofluorescence, Staining, Fluorescence, Control