Figures & data
Figure 1. Diosmetin impairs the genomic stability of EC cells and inhibits the cell proliferation
(a): Effects of diosmetin on the proliferation of ISK, Hec-1B, AN3CA cells. Cells were seeded in 6-well plates at 2 × 105 cells (ISK, Hec-1B) or 3 × 105 cells (AN3CA) per well and treated by DMSO or diosmetin in indicated concentration for 72 hours, followed by cell number counting. Error bars represent SD. (b): Representative pictures of alkaline comet assay. Hec-1B cells were incubated with DMSO or diosmetin in indicated concentration for 72 hours and subjected to the comet assay. Scale bar, 100 μM. (c): Statistical analysis of alkaline comet assay. The tail moments of at least 50 cells were quantified using the Cometscore software (Sumerduck, VA, USA). Results were presented as mean ± SEM. Mann-Whitney U Test was used to compare the difference. ****: P < 0.0001.
Figure 2. Diosmetin negatively regulates HR repair in EC cells
(a,b): Reporter constructs for DSB repair analysis. HR (a) and NHEJ (b) reporters are constructed and function as previously described [Citation10,Citation26]. (c,d): Analysis of relative HR (c) and NHEJ (d) efficiencies in cells treated by DMSO or diosmetin in indicated concentration. Hec-1B cells were pretreated with DMSO or diosmetin for 24 hours, followed by being transfected with I-SceI digested reporters along with DsRed plasmids. Transfected Hec-1B cells were continued to be treated with the above reagents until 72 hours in the incubator. Cells were collected and examined on the flowcytometry. The ratio of GFP+ versus DsRed+ cells was measured as relative HR or NHEJ efficiency. Students’ t test was used to compare the difference, *: P < 0.05, **: P < 0.01, NS: no significance. Error bars represent SD. (e): Representative pictures of EdU incorporation assay. Hec-1B cells were incubated with DMSO or diosmetin in indicated concentration for 72 hours and subjected to the EdU incorporation assay and then examined on flowcytometry. (f): Statistical analysis of EdU incorporation assay. The distribution of S phase was quantified on the FLOWJO software. At least 10,000 cells were examined. Students’ t test was used to compare the difference. NS: no significance. Error bars represent SD.
Figure 3. Diosmetin reduces the recruitment of RPA2
(a–c): Representative images of γH2AX (a), RPA2 (b), RAD51 (c) foci at different time points post IR (left) and statistical quantifications of the foci number (right). HCA2-hTERT cells were incubated with DMSO or diosmetin for 24 hours, then treated with X-ray (2 Gy). Cells were harvested immediately (0 h) or cultured for 2 h, 4 h, 16 h, 24 h before being harvested for immunofluorescence assay. Images were taken with a laser scanning confocal microscope (TCS SP8; Leica, Wetzlar, Germany). Scale bar, 10 μM. For each time point, at least 50 cells were counted. Error bars represent SEM.
Figure 4. Diosmetin sensitizes EC cells to ionizing radiation
(a): Representative pictures of clonogenic assay manifesting viability of Hec-1B cells after diosmetin or IR treatment or both. (b): Statistical analysis of clonogenic assay. Hec-1B cells were pretreated with DMSO or diosmetin in indicated concentration for 24 hours, followed by irradiation in indicated dose. Then cells were continued to be treated with DMSO or diosmetin for 72 hours and incubated in the incubator until 2 weeks. Colonies with at least 50 cells were qualified. Analysis of nonlinear regression was used. The IC50 values were analyzed using a dose-effect analysis software. Error bars represent SD.
Supplemental material
