FIGURE 7.

NPM1 alleviates ATF5-dependent CRE repression, blocks ATF5-dependent inhibition of cell proliferation and colony formation potential, and removes ATF5-induced G₂/M block in Hep3B cells. A, HEK293 cells were cotransfected with a reporter plasmid expressing luciferase under the control of the CRE sequence, Renilla control vector, RSV-Cα (expression vector for the catalytic subunit of PKA), and vectors empty (−) or expressing ATF5 (+), WT (NPM1) or C-terminal truncation of NPM1 (NPM1-c) at indicated DNA amount, and in the absence or presence of MG132 (20 μm) and BAF (50 μm) (MG+BAF). Luciferase activities were reported as relative units after correction with the Renilla activities. Expression of the transfected FLAG-ATF5, Myc-NPM1, and β-actin (loading control) was monitored by Western immunoblotting (IB) analysis (top panels). B, semi-quantitative RT-PCR analysis of PEPCK, c-IAP2, R-RAS, and YWHAH in response to ATF5 and NPM1 expression in Hep3B cells. β-Actin was used as control. C and D, expression of NPM1 reverses ATF5-induced inhibition of cell proliferation and survival in Hep3B cells. Hep3B cells were coinfected with a retrovirus empty (Control) or expressing NPM1 and a retrovirus empty (−) or expressing ATF5 (+). Cell viability and proliferation relative to vector control was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays 5 days after infection (C) and colony formation assay (D). *, p < 0.05; **, p < 0.01, two-tailed Student's test. E, Hep3B cells infected with retroviruses as described in C were stained with propidium iodide, and cell cycle distribution (%) was analyzed by flow cytometry.