Research Paper Volume 13, Issue 11 pp 15193—15213

TRDMT1 participates in the DNA damage repair of granulosa cells in premature ovarian failure

The oxidative DNA damage repair promoted by TRDMT1 is related to RNA m5C methylation. (A) ROS in CTX-KGN were detected by DHE probe after transfection with GFP-NC or GFP-TRDMT1 (WT, G155V, and E63K mutant). Scale bar: 100 μm. (B) The DNA damage of CTX-KGN transfected with GFP-NC or GFP-TRDMT1 (WT, G155V, and E63K mutant) was measured via comet assay. Scale bar: 100μm (C) CTX-KGN transfected with GFP-NC and GFP-TRDMT1 (WT, G155V, and E63K mutant) were stained for γ-H2AX at the indicated time points. Scale bar: 10μm. Positive cells in each group and the frequency of cells with foci were counted. (D) Apoptosis of CTX-KGN transfected with GFP-NC or GFP-TRDMT1 (WT, G155V, E63K mutant) was assessed using Annexin V staining. Comparison of apoptotic rates between the groups. (E) The percentage of m5C in total RNA from CTX-KGN after transfection with GFP-NC or GFP-TRDMT1 (WT, G155V, and E63K mutant) was detected via ELISA-based assays. (F) The expression of RAD51 and RAD52 were detected after we stably expressed TRDMTWT and the mutants (TRDMT1G155V/E63K) in TRDMT1 KD CTX-KGN. *P

Figure 5. The oxidative DNA damage repair promoted by TRDMT1 is related to RNA m5C methylation. (A) ROS in CTX-KGN were detected by DHE probe after transfection with GFP-NC or GFP-TRDMT1 (WT, G155V, and E63K mutant). Scale bar: 100 μm. (B) The DNA damage of CTX-KGN transfected with GFP-NC or GFP-TRDMT1 (WT, G155V, and E63K mutant) was measured via comet assay. Scale bar: 100μm (C) CTX-KGN transfected with GFP-NC and GFP-TRDMT1 (WT, G155V, and E63K mutant) were stained for γ-H2AX at the indicated time points. Scale bar: 10μm. Positive cells in each group and the frequency of cells with foci were counted. (D) Apoptosis of CTX-KGN transfected with GFP-NC or GFP-TRDMT1 (WT, G155V, E63K mutant) was assessed using Annexin V staining. Comparison of apoptotic rates between the groups. (E) The percentage of m5C in total RNA from CTX-KGN after transfection with GFP-NC or GFP-TRDMT1 (WT, G155V, and E63K mutant) was detected via ELISA-based assays. (F) The expression of RAD51 and RAD52 were detected after we stably expressed TRDMTWT and the mutants (TRDMT1G155V/E63K) in TRDMT1 KD CTX-KGN. *P<0.05, **p<0.01, and ***p<0.001. Statistical significance was determined using two-tailed t-tests for two groups and ANOVA for multiple comparisons. All values are means ± SD.