Fig. 2

CHIP overexpression did not affect cell growth of DLD-1 cells. a The cell growth curves of the hCHIP and ctrl cells were detected using a real-time x-Celligence system. E-plate was plated with 10,000 cells/well and the cell growth was continuous monitored for 72 h. b The apoptosis of the hCHIP and ctrl cells was determined by the AnnexinV/PI staining. AnnexinV positive and AnnexinV/PI positive cells were determined as apoptosis cells. Values ± SD from three individual experiments were displayed. c Cell proliferation of the hCHIP and ctrl cells were determined by the Brdu proliferation assay. 96-well plate was plated with 10,000 cells/well and was added 10 μl buffer after cultured for 24, 48 and 72 h. OD450 was measured by means of spectrophotometer microplate reader. d Cell cycle parameters of hCHIP and ctrl cells were determined by flow cytometry analysis using propidium iodide. The histograms report the percentage of cells in G0/G1, S and G2/M phases of all the establishing cell lines. e Western blot analysis of the protein expression of total ERK1/2, p-ERK1/2, p38, p-p38 as well as total AKT and p-AKT⁴⁷³ and p-AKT³⁰⁸ in hCHIP and ctrl cells. Actin was used as an internal control. f Western blot analysis of the protein expression of the NF-κB subunits. Actin was used as an internal control. g Western blot analysis of the protein expression of the cell cycle related proteins cyclinD1. Actin was used as an internal control