The expression of STAT3 proteins was evaluated in FaDu cells when treated by single or combinational administration of BBM and radiation.Notes: (A) Typical images of p-STAT3 and STAT3 protein by Western blot. (B) Semi-quantification of different protein expression. **P<0.01.Abbreviations: BBM, berbamine; Con, control; R, radiation.

Western blot detected the expression of p21, CDK1 and Bcl-2 in RA549 under 6 Gy treatment.

) Western blot detected the expression of p21, CDK1 and Bcl-2 in RA549 under 6 Gy treatment.

The TBARS level was checked after 24 h of irradiation. The cells were incuba

To determine DNA DSB repair, we used an anti-γH2AX to immunofluorescently stain γH2AX foci at different time-points after exposure to IR. As shown in Fig. 3A without exposure to IR, few γH2AX foci were observed in the three cell groups. After irradiation with 2 Gy IR, γH2AX foci rapidly increased. The numbers of γ-H2AX foci in the 3 groups were almost equal at 0.5 h after IR. However, foci in the LP-H1802Lv201 cells disappeared more quickly. In contrast to the control and LP-NegLv201 cells, γH2AX foci in the LP-H1802v201 cells were significantly decreased at 3, 6 and 24 h after IR. Cells having more than 10 γH2AX foci were scored as γH2AX-positive cells. We found that the percentage of γH2AX-positive cells was significantly decreased in the LP-H1802Lv201 cell group at 6 and 24 h after IR. In contrast, at 0.5 and 3 h after IR, the percentages of γH2AX-positive cells in the 3 groups did not differ significantly. We also assessed RAD51 foci at 6 h after IR or under the condition without IR. As shown in Fig. 3B, without IR, RAD51 foci did not show a significant difference in the 3 groups. At 6 h after IR, the number of RAD51 foci in the LP-H1802Lv201 cells was significantly less than that in the control and LP-NegLv201 cells. Cell having more than 10 RAD51 foci were scored as RAD51-positive cells. The percentage of RAD51-positive cells was also lower in the LP-H1802Lv201 cell group. Western blot analyses were also used to assess γH2AX and RAD51 expression at 24 h after IR or under the condition without IR (Fig. 3C). Without IR, expression levels of γH2AX and RAD51 in the 3 cell groups were almost equal. At 24 h after IR, expression levels of γH2AX and RAD51 were significantly increased in contrast to the condition without IR. However, expression levels of γH2AX and RAD51 were lower in the LP-H1802Lv201 cells. We concluded that IR caused DNA DSBs equally in the 3 cell groups. Yet, SHP-1-overexpressing cells showed an enhanced DSB repair capacity.

Figure 3. The correlation between the changes in ERCC1 expression level and radiation resistance. The cell extracts were collected from (A) HCT116-Tet-on-ERCC1 or (B) COLO205-Teton-ERCC1 cells treated with 0, 0.5, 1, or 2 μg/mL doxycycline for 24 h. Anti-ERCC1 or anti-β-actin mAbs were used to detect ERCC1 or β-actin. (C) COLO205-Tet-on and (D) HCT116-Tet-on cells were treated with 0, 0.5, 1, or 2 μg/mL doxycycline for 24 h following exposure to 8 Gy radiation for 48 h. Cell viability (%) = the luminescence units of the sample groups/the luminescence units of the control group mean × 100%. * indicates a significant difference, p < 0.05. The luminescence units were measured by multimode plate reader (VICTORTM X2, PerkinElmer).