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. 2011 Aug 24;6(8):e23887. doi: 10.1371/journal.pone.0023887

Figure 5. PKR regulation by 5-FU treatment in HCT116 p53+/+ and HCT116 p53−/− cells.

Figure 5

(A) HCT116 p53+/+, HCT116 p53−/− cells were mock-treated, treated with 500 IU/ml of human IFNα during 16 hours, or treated with 10 µM of 5-FU during 4, 8, 16 and 24 hours. Total proteins were extracted for immunoblot analysis using anti whole p53, PKR, anti phospho PKR, anti whole PKR, anti phospho eIF2α, anti whole eIF2α, anti cleaved caspase-3, and anti β-actin antibodies. (B) HCT116 p53+/+, HCT116 p53−/− cells were mock treated or treated with 10 µM of 5-FU or 500 IU/ml of IFNα during 16 hours. Expression of endogenous mRNA of PKR was analyzed by real-time RT-PCR (means±SEM, n = 3) as described in material and methods. (C) HCT116 p53+/+, HCT116 p53−/− cells expressing shRNA against PKR or control shRNA mock-treated, or treated with 10 µM of 5-FU during 48 hours. After treatment cells were trypsinized and analyzed by flow cytometry to AnnexinV positive determination. Total proteins of both cell lines were extracted for immunoblot analysis using anti whole PKR and β-actin antibodies. The asterisks “**” designates p<0.01 in HCT116 p53+/+ cells expressing shRNA against PKR versus cells expressing the control shRNA. The asterisk “*” designates p<0.05 in HCT116 p53−/− cells expressing shRNA against PKR versus cells expressing the control shRNA.