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.
Question
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.
Solution
The above passage describes an experiment to determine DNA double-strand break (DSB) repair using an anti-γH2AX to stain γH2AX foci at different time-points after exposure to ionizing radiation (IR).
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Initially, without exposure to IR, few γH2AX foci were observed in the three cell groups.
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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.
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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.
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Cells having more than 10 γH2AX foci were scored as γH2AX-positive cells. The percentage of γH2AX-positive cells was significantly decreased in the LP-H1802Lv201 cell group at 6 and 24 h after IR.
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RAD51 foci were also assessed at 6 h after IR or under the condition without IR. 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.
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Western blot analyses were also used to assess γH2AX and RAD51 expression at 24 h after IR or under the condition without IR. 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.
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The conclusion drawn from the experiment is that IR caused DNA DSBs equally in the 3 cell groups. Yet, SHP-1-overexpressing cells showed an enhanced DSB repair capacity.
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