Figure 5. Zhangfei suppresses cell growth and UPR in wild-type p53-expressing U2OS cells, but not in p53-null MG63 cells. (A) p53-dependent transactivation in U2OS and MG63 cells. Cells were transfected with 0.5 µg of p53 reporter (pCAT3B-p53RE) for 24 h, then p53 transcriptional activity was determined by CAT ELISA assay. (B) U2OS and MG63 cells express Zhangfei when infected with adenovirus vector expressing the protein. Cells were infected with Adeno-ZF, and 24 h later Zhangfei protein was visualized by immunofluorescence (bar = 10 µm). (C) Ectopic expression of ZF suppresses cell growth in p53-wild type U2OS cells, but has no effect in p53-null MG63 cells. U2OS and MG63 human osteosarcoma cells were mock-infected or infected with Adeno-ZF or Adeno-LacZ and their growth rates were assessed at different time points after infection as absorbance at 405 nm after treatment with WST-1. (D) Zhangfei negatively regulates the UPR in U2OS cells but not in MG63 cells. U2OS and MG63 human cells were either mock-infected or infected with Adeno-ZF or Adeno-LacZ. Twenty-four hours after infection, cells were treated with thapsigargin for 4 h, and then harvested. The differences in levels of transcripts for UPR genes (Xbp1s, CHOP, and GRP78) were determined by qRT-PCR. (E) Zhangfei enhances p53-dependent transcripts in U2OS cells but not in MG63 cells. Transcripts for p53, p21, PIG3, and NOTCH1 in U2OS and MG63 cells either mock-infected or infected with Adeno-LacZ or Adeno-ZF were measured by pRT-PCR. (F) Zhangfei increases p53 and p21 proteins in U2OS cells but not in MG63 cells. p53 and p21 proteins were detected by immunoblotting in U2OS and MG63 cells either mock-infected or infected with Adeno-ZF. The values in (D and E) represented fold changes of transcripts between Adeno-LacZ (white bar) or Adeno-ZF (black bar) infected cells with mock-infected cells. The means and standard deviations of experiments (n = 3) were shown. P < 0.05 were considered to be significant. Error bars indicate standard deviations from means. Horizontal dotted line indicated a 2-fold change. A change of more than 2-fold was arbitrarily regarded as significant.