Figure 3.

Effects of combined HSPA1 and HSPA2 deficiency on the proliferation, clone-forming ability and chemoresistance of NCI-H1299 cells. (A) Levels of HSPAs and HSPC in wild-type (wt); control sh-luc/sh-luc cells doubly transduced with a non-targeting shRNA-luc sequence; sh-A1.S/sh-A2.4 cell line simultaneously transduced with HSPA1- and HSPA2-targeting sequences. Representative immunoblots are shown (n = 3), actin was used as a protein loading control. (B) Cell proliferation assessed using MTS assay. Results are expressed as mean ± SD (n = 5, each in three technical replicas) in relation to values obtained at 24 h after plating. (C) Results of crystal violet staining proliferation assay (n = 4, each in three technical replicas). Relative absorbance of stained cells was plotted against time (24–72 h) of continuous growth. (D) Number of colonies formed by cells plated onto 6-well dishes (1 × 10³ cells/well) and cultured for 7–8 days. Colonies were counted manually (mean ± SD, n = 7 each in three technical replicas). (E,F) Effects of simultaneous HSPA1 and HSPA2 deficiency on resistance of cells to cisplatin (CDDP) (E) or bortezomib (BTZ) (F). Cell viability was measured using MTS assay after 72 h treatment. Results are expressed relative to untreated control (mean ± SD, n = 4, each in triplicate, *p < 0.05, statistical significance was determined by two-tailed t-test).