Research Paper Volume 13, Issue 12 pp 15833—15874

The lack of functional DNMT2/TRDMT1 gene modulates cancer cell responses during drug-induced senescence

DNMT2/TRDMT1 gene knockout in four cancer cell lines, namely HeLa cervical cancer cells, MDA-MB-231 breast cancer cells, U-2 OS osteosarcoma cells and U-251 MG glioblastoma cells (A) and DNMT2/TRDMT1 gene knockout-mediated changes in the cell cycle of DOX- and ETOPO-treated cancer cells (B). (A) Western blot-based analysis of the protein levels of DNMT2/TRDMT1. Anti-β-actin antibody served as a loading control. (B) DNA content-based analysis of cell cycle was conducted using flow cytometry. Bars indicate SD, n = 3, ***p a posteriori test), ###pa posteriori test). CTR, control conditions; DOX, doxorubicin treatment; ETOPO, etoposide treatment; AZA, azacytidine treatment; MIX, azacytidine post-treatment; C-NIC, control cells with unmodified levels of DNMT2/TRDMT1 containing control plasmid; D-NIC, cells with DNMT2/TRDMT1 gene knockout containing dedicated DNMT2 double nickase plasmid.

Figure 1. DNMT2/TRDMT1 gene knockout in four cancer cell lines, namely HeLa cervical cancer cells, MDA-MB-231 breast cancer cells, U-2 OS osteosarcoma cells and U-251 MG glioblastoma cells (A) and DNMT2/TRDMT1 gene knockout-mediated changes in the cell cycle of DOX- and ETOPO-treated cancer cells (B). (A) Western blot-based analysis of the protein levels of DNMT2/TRDMT1. Anti-β-actin antibody served as a loading control. (B) DNA content-based analysis of cell cycle was conducted using flow cytometry. Bars indicate SD, n = 3, ***p < 0.001 compared to CTR (ANOVA and Dunnett’s a posteriori test), ###p<0.001 compared to drug-treated C-NIC cells (ANOVA and Tukey’s a posteriori test). CTR, control conditions; DOX, doxorubicin treatment; ETOPO, etoposide treatment; AZA, azacytidine treatment; MIX, azacytidine post-treatment; C-NIC, control cells with unmodified levels of DNMT2/TRDMT1 containing control plasmid; D-NIC, cells with DNMT2/TRDMT1 gene knockout containing dedicated DNMT2 double nickase plasmid.