dnmt1 and uhrf1 dna fragments  (GenScript corporation)

Journal: Cell Communication and Signaling : CCS

Article Title: UHRF1 phase separation mediates stable inheritance of DNA methylation and promotes the proliferation of prostate cancer cells

doi: 10.1186/s12964-025-02309-6

Figure Lengend Snippet: UHRF1 expression is negatively correlated with the DNA methylation level at the promoter of silenced genes in prostate cancer (PC) cells. A Heatmap displaying gene expression of PC tissues and normal prostate tissues (right panel) and methylation levels in the promoter regions (left panel). Gene expression levels are shown as log₂-transformed transcripts per million (TPM). Methylation levels were calculated using the log 2 transformation of fragments per kilobase of transcript per million mapped reads (FPKM) within ± 1.5 kb of the transcription start site (TSS), derived from whole genome bisulfite sequencing ( GSE104789 ). B mRNA expression of frequently methylated genes ( GSTP1 , RASSF1 , and TIMP2 ) in C4-2B cells after transfection with control shRNA (sh-CTRL) or shRNA-UHRF1 (sh-UHRF1) by real-time quantitative PCR (RT-qPCR) ( n = 3). The experiment was repeated three times. C GSTP1, RASSF1, and TIMP2 protein levels in C4-2B cells transfected with sh-CTRL or sh-UHRF1 by Western blot. The experiment was repeated three times. D mRNA expression of high frequently methylated genes ( GSTP1 , RASSF1 , and TIMP2 ) in DU145 cells after transfection with sh-CTRL or sh-UHRF1 by RT-qPCR ( n = 3). The experiment was repeated three times. E GSTP1, RASSF1, and TIMP2 protein levels in DU145 cells transfected with sh-CTRL or sh-UHRF1 by Western blot. The experiment was repeated three times. F Bisulfite sequencing methylation patterns at GSTP1 and RASSF1 sequences in C4-2B and DU145 cells. CG dinucleotides are represented by circles, solid if methylated and empty if unmethylated. Percentages of methylated CG dinucleotides are shown below each pattern. G , H DNMT1 protein expression in C4-2B and DU145 cells after transfection with sh-CTRL or sh-UHRF1 by Western blot. The experiment was repeated three times. I , J DNMT1 mRNA in PC cells after transfection with sh-CTRL or sh-UHRF1 by RT-qPCR ( n = 3). The experiment was repeated three times. K UHRF1 formed nuclear condensates and was colocalized with DNMT1 in C4-2B cells. Cells were transfected with UHRF1-EGFP and DNMT1-mCherry for 48 h and imaged. mCherry and EGFP were used as controls. Data are presented as mean ± SEM by two-tailed unpaired t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant

Article Snippet: The DNMT1 and UHRF1 DNA fragments found in this study were produced by Genscript and amplified by polymerase chain reaction (PCR) using Phanta ® Max Super-Fidelity DNA Polymerase (Catalog #P505-d1, Vazyme, Nanjing, China).

Techniques: Expressing, DNA Methylation Assay, Gene Expression, Methylation, Transformation Assay, Derivative Assay, Methylation Sequencing, Transfection, Control, shRNA, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: UHRF1 phase separation mediates stable inheritance of DNA methylation and promotes the proliferation of prostate cancer cells

doi: 10.1186/s12964-025-02309-6

Figure Lengend Snippet: TSGs expression and DNMT1 localization are altered when the phase separation of UHRF1 is disrupted. A 1,6-hex disrupted droplet formation of UHRF1 that reduced the interaction between UHRF1 and DNMT1. Cells transfected with EGFP control or EGFP-tagged UHRF1 were subjected to immunoprecipitation with DNMT1 antibody. The presence of UHRF1 in the immunoprecipitate was assessed by Western blotting against an anti-GFP antibody. The experiment was repeated three times. B Enrichment of DNMT1 within ± 1.5 kb from the transcription start sites (TSSs) of all genes in the 1,6-hex treated group and control. IgG signal was used as a negative control. Genes are sorted by their CUT&Tag signal, and profiles are shown as heatmaps. C DNMT1 enrichment in all cytosine-phosphate-guanine (CpG) island regions in 1,6-hex treated (1.5% for 30 min) group and the control. IgG signal was used as a negative control. Regions were sorted by their CUT&Tag signal, and profiles are shown as heatmaps. D , E Statistical analysis of CUT&Tag data (using antibody against DNMT1, n = 3). F GSTP1 and RASSF1 mRNA expression levels in PC cells after 1,6-hex treatment (0.5% for 8 h). The experiment was repeated three times. G Bisulfite sequencing methylation patterns at the GSTP1 and RASSF1 sequences in PC cells after 1,6-hex treatment (0.5% for 8 h). The percentages of methylated CG dinucleotides are displayed beneath each pattern, with CG dinucleotides represented by circles that are solid if methylated and empty if unmethylated. Data are presented as mean ± SEM by two-tailed unpaired t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant

Article Snippet: The DNMT1 and UHRF1 DNA fragments found in this study were produced by Genscript and amplified by polymerase chain reaction (PCR) using Phanta ® Max Super-Fidelity DNA Polymerase (Catalog #P505-d1, Vazyme, Nanjing, China).

Techniques: Expressing, Transfection, Control, Immunoprecipitation, Western Blot, Negative Control, Methylation Sequencing, Methylation, Two Tailed Test