PMC

Cells were seeded in a 12-well plate at a density of 1×104 cells per well. After 24 h, cells were collected and counted on day 1 to day 8, respectively. Cell number was obtained from triplicates. Then growth rate of cells was calculated. No significant difference was observed among the three cell lines (P>0.05).

The cells were treated by 0, 0.05, 0.5 mg/ml MMC for 24 h. Micronucleus rate (%) was calculated as the ratio of micronucleated cells in total cells examined. The data were analyzed by Poisson distribution. “a” denotes a significant difference (P<0.05) between MMC treated cells and untreated control. “b” indicates a significant difference (P<0.05) between pol β^−/− or pol β^oe cells and pol β^+/+ cells.

The cells were treated by increasing dosages of MMS for 24 h. The hprt gene mutation frequency (%) was calculated by the equation described in Section 2 and was plotted against the concentration of MMS. Poisson distribution was used to determine significant differences. “a” denotes a significant difference (P<0.05) between treated groups and untreated control, whereas “b” indicates a significant difference (P<0.05) between pol β^−/− or pol β^oe cells and pol β^+/+ cells.

Total cell lysate was prepared in sodium dodecyl sulfate (SDS) buffer. Same amount of total proteins isolated from pol β^−/−, pol β ^+/+ and pol β^oe cells were separated by 6% SDS-polyacrylamide gel electropheresis and transferred onto a polyvinylidene fluoride (PVDF) membrane. After incubation with the primary polyclonal antibody against pol β, the PVDF membrane was incubated with a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase. Pol β protein was visualized with enhanced chemiluminescence. Western blot of actin protein was used as a loading control.

Cells survival responses to MMS at various dosages were examined. (A) Cell viability was measured using MTT assay that was performed according to the description of Section 2 and was plotted against the concentration of MMS. (B) Cells proliferative capacity was detected by colony forming assay. Cells were exposed to 0, 0.01, 0.05, 0.1, 0.25, 0.5 mM MMS. The rate of colony forming (%) was calculated according to the equation provided in Section 2. For both assays, data were illustrated as mean ± standard error derived from three independent assays. One-way analysis of variance (ANOVA) including least-significant difference (LSD) multiple comparison test was employed to determine statistical differences, P<0.05 was considered to be statistically significant.

Comet assay was used to detect cellular K₂Cr₂O_7-induced DNA strand breaks under various expression levels of pol β. (A) The comet rate (%) resulting from pol β^−/−, pol β^+/+ and pol β^oe cells was calculated according to the equation provided in Section 2 and was plotted against various concentrations of K₂Cr₂O₇. 200 cells were scored per slide. (B) The OTM values were expressed as mean ± S.D. and obtained from 30 comet images for each slide. For both of comet rate and OTM, One-way analysis of variance (ANOVA) including least-significant difference (LSD) multiple comparison test was used to identify statistically significant difference. “a” denotes a significant difference (P<0.05) between K₂Cr₂O₇-treated cells and untreated cell control, “b” indicates a significant difference (P<0.05) between pol β^−/− or pol β^oe cells and pol β^+/+ at the same concentration of. K₂Cr₂O₇.