Figure 1.

ABTL0812 induces ER stress in cancer cell lines. (a, b) ABTL0812 induces dynamic autophagy. Cells were preincubated 3 h with vehicle or lysosomal protease inhibitors E64d (10 µmol/L) and pepstatin A (PA, 10 µg/mL) (a) or with inhibitor (50 nM) of the vacuolar-type ATPase, bafilomycin A₁ (BafA) (b) before treatment with ABTL0812 for 24 h. Levels of lipidated and non-lipidated MAP1LC3B proteins were monitored by immunoblotting. (c) ABTL0812 induces autophagy-mediated cancer cell death. Effect of ABTL0812 treatment (48 h) in viability of MiaPaca2 or A459 stable cell lines transfected with control shRNA (shC) or ATG5-selective shRNA (shATG5). Right panels show the corresponding immunoblots. (d) AKT or MTOR inhibition does not result in the induction of autophagy. MiaPaca2 cells were treated with 25 µmol/L AZD5363 (AKT inhibitor), 0.1 µmol/L AZD2014 (MTOR inhibitor), 10 µmol/L everolimus (MTORC1 inhibitor) or 100 µmol/L ABTL0812 for 24 h. Expression of RPS6, p-RPS6, lipidated and non-lipidated MAP1LC3B and ACTB proteins were determined by immunoblotting. Similar results were obtained in four separate experiments. (e–h) ABTL0812 induces ER stress in cancer cells. (e) Cells were treated with 100 µmol/L ABTL0812 or 200 nmol/L Brefeldin-A (BrefA), total RNA isolated and cDNA synthesized by RT-PCR. XBP1 splicing was determined by PCR using primers that amplify both spliced (XBP1 s) and unspliced (XBP1 u) mRNA species. Similar results were obtained in four separate experiments. (f) MiaPaca2 and A549 cells were treated with 100 µmol/L ABTL0812, then lysed. The levels of p-EIF2A were monitored by immunoblotting. Similar results were obtained in three separate experiments. (g) Cells were treated with 100 µmol/L ABTL0812, then lysed. The levels of ER stress markers HSPA5, ATF4, DDIT3 and TRIB3 were monitored by immunoblotting. The marker of autophagy MAP1LC3B was also analyzed. Similar results were obtained in four separate experiments. (h) MiaPaca2 and A549 cells were treated with 100 µmol/L ABTL0812 for 24 h, pelleted, total RNA isolated, and ATF4, DDIT3 and TRIB3 mRNA levels were analyzed by RT-qPCR. Each value is the mean ± SD of three different experiments. **, P < 0.005; ***, P < 0.001, Student’s t-test