Figure 4.

DDIAS level is regulated by CHIP expression in lung cancer cells. (a) Expression of CHIP and DDIAS in lung cell lines. Normal lung fibroblast cells, WI-38 and CCD-34 LU, and lung cancer cells, NCI-H460, HCC-827, NCI-H1299, NCI-H1437 and NCI-H1703, were used. Cell lysates of indicated cell lines were immunoblotted to detect expression of DDIAS, CHIP, HSP70 and HSP90. (b) CHIP overexpression decreased endogenous DDIAS protein level in NSCLC. H1299 and H1703 cells were transfected with HA-CHIP for 48 h and lysed. Endogenous DDIAS protein level was examined using anti-DDIAS, anti-HA and anti-GAPDH. (c) Stabilities of DDIAS by HA-CHIP overexpression in NCI-H1299. DDIAS stability was determined in HA-CHIP-overexpressed NCI-H1299 cells in the presence of CHX (20 μM). (d) The CHIP knockdown effect on DDIAS stability in NCI-H1437 cells. DDIAS stability was determined in cells transfected with CHIP siRNA. (e) The effect of CHIP overexpression on the growth of NCI-H1299 and NCI-H1703 cells. Growth inhibition by CHIP overexpression was determined by an SRB assay. The growth of NCI-H1299 or NCI-H1703 cells transiently expressing HA-CHIP or CHIP mutants was also compared. The values represent the mean ± SEM of three independent experiments with triplicated measurements. *P<0.05, ***P<0.005 vs Con. (f) The effect of Flag-DDIAS overexpression on the growth of NCI-H1299 and NCI-H1703 cells. The effect of Flag-DDIAS overexpression on the growth inhibition by HA-CHIP overexpression was determined by an SRB assay. The values represent the mean ± SEM of three independent experiments with triplicate measurements. **P<0.01 vs Con. (g) Western blot analysis of cells overexpressing FLAG-DDIAS and/or HA-CHIP