Fig. 5

PCBP1 up-regulates p27 expression by enhancing the mRNA stability and translation. (a) Immunoblot of endogenous p27 protein levels upon transcription inhibition or/and proteasome degradation repression. A2780 cells overexpressing GFP-PCBP1 or GFP alone were treated with DMSO, Act D at 0.5 μg/ml for 8 h, MG132 at 20 μM for 4 h, or with their combination for 4 h, and analyzed. Results are representative of at least three independent experiments. (b) Immunoblot of endogenous p27 protein levels in A2780 cells with endogenous PCBP1 knockdown. Cells were treated with MG132 as in (a) (c) Immunoblot of p27 ubiquitin ligase Skp2 and p27 expression in the indicated cells. No evident Skp2 expression alteration is shown. (d) Immunoblot of the indicated proteins possibly related to p27 protein expression and cell survival. (e) Semi-quantitative RT-PCR analysis of p27 or c-myc mRNA stability in A2780 cells overexpressing PCBP1 or GFP control, on condition of Act D treatment to terminate the novel mRNA transcription. c-myc and GAPDH were used as positive and negative controls. The PCR of p27 were performed with 20 cycles, c-Myc with 25 and GAPDH for 20 cycles, respectively. Relative p27 and c-myc level against GAPDH were further normalized against DMSO treated GFP and shown at the bottom. (f) Semi-quantitative RT-PCR analyses of p27 or c-myc mRNA stability in A2780 cells with endogenous PCBP1 knockdown by specific shRNAs as the method in E. *p<0.05 (g, h) Analyses of p27 or c-myc mRNA duration in the cells as shown in (e, f) Band intensities were measured by ImageJ software and normalized against their corresponding GFP-Lane treated with DMSO. All experiments were independently repeated at least for 3 times. **p<0.01 (I) Ribosome profiling of p27 mRNA. Major percentage of p27 mRNA cells is located in the lighter polysome fractions (1–8) of the parental DLD-1, while overexpression of PCPB1 results in a shift of p27 mRNA to the heaver fractions (3–10). Beta-actin mRNA is shown as an internal negative control. The marked numbers are equivalent to the collected fractions after gradient centrifuge. Fraction 1 represents the top (10%) of the gradient, fraction 10, the bottom (50%) of the gradient