Figure 3.

Involvement of reactive oxygen species in senescence induced by gemcitabine and ionizing radiation.

IOMM-Lee and HKBMM cells plated on 6-well plates (2 × 10⁵ for IOMM-Lee and 4 × 10⁵ for HKBMM per each well) were incubated without or with gemcitabine (GEM, 3 nM for IOMM-Lee, and 2 nM for HKBMM) in the absence or presence of N-acetyl-cysteine (NAC, 5 mM) for 4 days and not irradiated or irradiated twice by X-ray (IR, 1 Gy for IOMM-Lee and 2 Gy for HKBMM) on days 1 and 3, and then subjected to a CellRox assay (A), cell viability assay (B), SA-β-gal staining (C), or γH2AX immunocytochemistry (D) on day 4. In (A), (B), and (C), experiments were performed in 4, 6, and 4 replicates, respectively, and values are shown as mean ± SD. In (D), the number of γH2AX foci was counted in more than 60 cells per group and shown as violin plots (line, median; dotted lines, quartile). P-values were calculated by a 1-way ANOVA with Tukey’s post hoc tests (A, B, and C) or by the Kruskal-Wallis test with Dunn’s multiple comparisons test (D). *P < .05. ^†P < .05 vs the Control (no treatment).