Figure 3.

HSPA5 has a pivotal role in NTS-mediated regulation of lysosomal activity. (A) FaDu cells were seeded in a 48-well plate and treated with NTS for the indicated times. Cells viability was measured by the MTT assay (n = 6). Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05). (B) FaDu cells were treated with or without NTS for 24 h in the absence of serum and lysosomal activity was measured by FACS analysis (n = 3). Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05) (*, vs O H). (C) GFP-MAP1LC3-II puncta was accumulated by inhibiting lysosomes. NH₄Cl (10 mM), E-64 (10 μM), and NTS were added to GFP-MAP1LC3-II-transfected cells for 24 h in the absence of serum. GFP-MAP1LC3-II puncta were observed by fluorescence microscopy. Scale bar: 20 μm. (D) HSPA5 overexpression inhibits NTS-induced downregulation of lysosome-related proteins or gene expression. FaDu cells were transfected with or without pcDNA3.1-HSPA5 for 24 h, and then cells were treated with NTS for the indicated times. The indicated proteins or gene expression were determined by western blot assay or RT-PCR. (E to G) HSPA5 overexpression inhibits NTS-induced downregulation of lysosomal activity. HSPA5-transfected or nontransfected cells were treated with NTS for 24 h and then lysosomal activity was assessed with FACS analysis (E), TFEB transcription factor activity assay (F) or CTSD (cathepsin D) activity assay (G). (E, F and G) Data are means ± SD. Asterisks indicate statistically significant differences (n = 3, P < 0.05).