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. 2015 Feb 19;6(6):4110–4125. doi: 10.18632/oncotarget.2893

Figure 7. Reduced CDDP uptake into the nucleus, CDDP-DNA adduct formation and chk1-p53 activation in p22phox stable lines.

Figure 7

(A) CDDP distribution and localization was monitored by Alexa Fluor 488 fluorescence in p22phox stable lines and p22phox-overexpressing KB cells using confocal microscopy. Magnification: 1000X. The respective cytoplasmic and nuclear fluorescence intensity in each cell was measured by Olympus Fluoview Viewer (Ver. 3.0 software). The average cytoplasm-to-nucleus intensity ratios were determined from the randomly selected 24, 20, 21, 21 and 12 cells in control line, p22phox stable line #1, p22phox stable line #2, mock transfected KB and p22phox-DsRed transfected KB, respectively. The quantitative results and statistical analysis were shown in the right panels. (B) The p22phox stable lines and the control line were treated with 10 or 20 μM CDDP overnight, and genomic DNA was isolated and analyzed by dot blot assay using anti-CDDP adducts antibody. The arrows denote non-specific background signals. (C) The cells were treated with CDDP (20 μM) for 0, 2, 4 or 6 h and the lysates were analyzed by Western blot analysis using antibodies against t-chk1, p-chk1, t-p53 and p-p53. The numbers below the blots were quantitative ratios of p-chk1, t-chk1, p-p53 or t-p53/GAPDH band intensities normalized to those without CDDP treatment. Abbreviations: c, cytoplasm; n, nucleus; pos, positive control. The experiments were repeated four times, and the representative images or data are shown.