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-mediated changes in the levels of senescence-associated-beta-galactosidase (SA-β-gal) positive cells (A, B) and nuclear p21 pools (C, D) in four cancer cell lines treated with DOX or ETOPO. (A, B) Cellular senescence was revealed using imaging cytometry. SA-β-gal activity is presented as relative fluorescence units (RFU). (B) Representative microphotographs are shown, objective 20x, nucleus staining (blue), SA-β-gal staining (green). (C, D) Nuclear p21 immuno-staining. (C) Nuclear p21 levels are presented as relative fluorescence units (RFU). (D) Representative microphotographs are shown, objective 20x, nucleus staining (blue), nuclear p21 immuno-staining (red), RESPONSE, representative DOX or ETOPO treatment. (A, C) Box and whisker plots are shown, 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 2. DNMT2/TRDMT1 gene knockout-mediated changes in the levels of senescence-associated-beta-galactosidase (SA-β-gal) positive cells (A, B) and nuclear p21 pools (C, D) in four cancer cell lines treated with DOX or ETOPO. (A, B) Cellular senescence was revealed using imaging cytometry. SA-β-gal activity is presented as relative fluorescence units (RFU). (B) Representative microphotographs are shown, objective 20x, nucleus staining (blue), SA-β-gal staining (green). (C, D) Nuclear p21 immuno-staining. (C) Nuclear p21 levels are presented as relative fluorescence units (RFU). (D) Representative microphotographs are shown, objective 20x, nucleus staining (blue), nuclear p21 immuno-staining (red), RESPONSE, representative DOX or ETOPO treatment. (A, C) Box and whisker plots are shown, n = 3, ***p < 0.001 compared to CTR (ANOVA and Dunnett’s a posteriori test), ###p<0.001 compared to C-NIC cells at the same culture conditions (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.