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. 2018 Oct 17;9:1197. doi: 10.3389/fphar.2018.01197

FIGURE 4.

FIGURE 4

CDDP selectively increased NER proteins in GC cells. (A) AGS and MKN45 cells were treated with 10 μg/ml CDDP and harvested at the indicated times. RT-qPCR was used to quantify the mRNA levels of XPA, XPC, XPF, XPG, and ERCC1 by using specific primers (see section “Materials and Methods”). The graphs represent the relative levels of each gene using ΔΔCT referred to the levels on a control no tumorigenic cell line HACAT, and using GAPDH as endogenous control. (B) XPC, XPD, XPA, and ERCC1 protein levels were detected by western blot in AGS and MKN45 cells after CDDP (10 μg/ml) treatment at the indicated times. α-Tubulin was used as a loading control. (C) XPA levels in AGS and MKN45 cells after 6 h of CDDP treatment (10 μg/ml) in the presence of caffeine (80 mM) and transfected with ATRWT or ATRDN plasmids. Arrow in XPA band indicates the non-processed protein. (D) Nuclear and cytoplasmic localization of XPC, XPD, and XPA proteins were detected by western blot in AGS and MKN45 cells after 6 h of CDDP treatment (10 μg/ml) and cellular fractioning (C, Cytoplasm; N, Nuclei). α-Tubulin was used as cytoplasmic protein loading control and lamin B1 was used as nuclear protein control. The experiments were repeated three times with similar results.