Figure 2.

NTS-induced inhibition of HSPA5 expression and its pivotal role in ER stress or autophagy. (A) FaDu cells were treated with NTS for the indicated times and protein levels were evaluated by western blot assay. (B) Inhibition of NTS-induced ER stress prevents autophagy. GFP-MAP1LC3-II plasmids were transfected into FaDu cells and 24 h later, the cells were pretreated with TUDCA (1 mg/ml) for 1 h. NTS treatment was given for 24 h with or without TUDCA in absence of serum. GFP-MAP1LC3-II puncta were analyzed with a fluorescence microscope (scale bar: 50 μm). (C) HSPA5 was decreased in response to NTS. FaDu cells were treated with NTS for 24 h in the absence of serum, and HSPA5 expression was determined by western blot assay (n = 3). (D) HSPA5 overexpression in HNC tissues. Proteins were isolated from frozen tissues of 6 patients with HNC, and HSPA5 expression level was determined by western blot assay (n = 6; C, cancer tissue; N, normal tissue; P, patient). (E) The immunohistochemistry analysis of HSPA5 in cancer or normal (scale bar: 200 μm). (F and G) HSPA5 overexpression inhibited NTS-induced ER stress, autophagy and cytotoxicity. HSPA5 plasmids were transfected into FaDu cells, and the cells were treated with NTS for 24 h in the absence of serum. Protein levels were evaluated by western blot assay (F) and MTT assay (G; n = 6). (C, D and G) Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05).