Figures & data
Figure 1 Differential DNMT1 expression in endometrial cancer cell lines.
Notes: (A) Upregulation of DNMT1 mRNA expression in poorly differentiated EC cell line (AN3CA and HEC-1B) compared to well-differentiated EC cell line (Ishikawa). (B) Western blot analysis of DNMT1 expression in various EC cell lines. GAPDH was the loading control. Shown are the representative blots.
Abbreviations: EC, endometrial carcinoma; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Abbreviations: EC, endometrial carcinoma; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 2 DNMT1 knockdown inhibits the proliferation of EC cells.
Notes: AN3CA and HEC-1B cells were transfected with DNMT1 siRNA or siRNA NC or untransfected. (A) qPCR analysis of DNMT1 expression at mRNA level in AN3CA cells. (B) qPCR analysis of DNMT1 expression at mRNA level in HEC-1B cells. (C) Western blot analysis of DNMT1 protein level in AN3CA cells. GAPDH was the loading control. Shown are the representative blots. (D) Western blot analysis of DNMT1 protein level in HEC-1B cells. GAPDH was the loading control. Shown are the representative blots. CCK-8 assay of the proliferation of AN3CA (E) and HEC-1B (F) cells after transfection with DNMT1 siRNA or siRNA NC. Data are represented as mean ± SD. **P<0.01 and ***P<0.001.
Abbreviations: CCK-8, Cell Counting Kit-8; EC, endometrial carcinoma; Con, control; NC, negative control; OD, optical density; ISK, Ishikawa; PCR, polymerase chain reaction; qPCR, real-time quantitative PCR.
Abbreviations: CCK-8, Cell Counting Kit-8; EC, endometrial carcinoma; Con, control; NC, negative control; OD, optical density; ISK, Ishikawa; PCR, polymerase chain reaction; qPCR, real-time quantitative PCR.
Figure 3 DNMT1 knockdown induces cell cycle arrest and apoptosis of AN3CA cells.
Notes: (A) Knockdown of DNMT1 induced cell cycle arrest in G0/G1 phase at 5 days after the transfection of DNMT1 siRNA. (B) Percentage of cells at G0 + G1, and S phases. The data presented have been background subtracted using data from unstained cells. (C) Flow cytometry analysis of AN3CA cells transfected with DNMT1 siRNA or siRNA NC. The lower right quadrants of the histograms indicate the percentage of early apoptotic cells. (D) Percentage of apoptotic cells. The data presented have been background subtracted using data from unstained cells.
Abbreviations: PI, propidium iodide; FITC, fluorescein isothiocyanate; NC, negative control; Ph, phase.
Abbreviations: PI, propidium iodide; FITC, fluorescein isothiocyanate; NC, negative control; Ph, phase.
Figure 4 DNMT1 knockdown alters the expression of apoptosis-related proteins.
Notes: qPCR (A) and Western blot (B) analyses of NF-κBIA and p65 mRNA and protein levels in AN3CA cells transfected with DNMT1 siRNA or siRNA NC. (C) Western blot analysis of Caspase-2 protein level in AN3CA cells transfected with DNMT1 siRNA or siRNA NC. qPCR (D) and Western blot (E) analyses of Bax and Bcl-2 mRNA and protein levels in AN3CA cells transfected with DNMT1 siRNA or siRNA NC. GAPDH was the loading control. Shown are the representative blots.
Abbreviations: NF-κBIA, nuclear factor kappa-B-inhibitor alpha; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; PCR, polymerase chain reaction; qPCR, real-time quantitative PCR.
Abbreviations: NF-κBIA, nuclear factor kappa-B-inhibitor alpha; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; PCR, polymerase chain reaction; qPCR, real-time quantitative PCR.
Figure 5 DNMT1 knockdown downregulates CCNDs.
Notes: (A) mRNA levels of CCND1 and CCND2 were measured by qPCR in AN3CA cells transfected with DNMT1 siRNA or siRNA NC. (B) Western blot analysis of CCND1 and CCND2 protein levels in AN3CA cells transfected with DNMT1 siRNA or siRNA NC. GAPDH was the loading control. Shown are the representative blots. Abbreviations: CCND1, cyclin D1; CCND2, cyclin D2; PCR, polymerase chain reaction; qPCR, real-time quantitative PCR; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
