Expression of CCKBR in gastric adenocarcinoma cells. AGS cells have low abundance of the CCKBR, MKN45 express the CCKBR endogenously and AGS-Gr cells are stably transfected with the CCKBR. S1b. Negative control images of the MKN45 cells stained for the CCKBR (phase contrast, Alexa 488, Draq5). Figure S2: Gastrin induces autophagy. AGS-Gr (a & b) cells treated with gastrin (10 nM), BafA1 (100 nM) and gastrin + BafA1 for 2 and 4 h. Protein expression of MAP1LC3B-II and SQSTM1 was analyzed by immunoblotting. The images shown represent one of three independent experiments. Graphs show mean +/- SEM (P- values: *** ≤ 0.01 **≤ 0.02, and * ≤ 0.05). Figure S3: Negative controls (primary antibodies omitted) for MAP1LC3B (Alexa 488) and SQSTM1 (Alexa 647). Figure S4: Gastrin mediated survival is dependent on autophagy. (a): A representative cytometric plots showing AGS-Gr cells treated with BafA1 and gastrin for 18 h. Cell viability was assessed using annexin V-PI staining and flow cytometric analyses. Blocking autophagy reduces gastrin mediated survival in AGS-Gr cells. (b): Cell viability assessed in AGS-Gr cells treated with gastrin (10 nM) for 6- 72 h. (c & d): Cells treated with gastrin (2 h) and subsequently treated with increasing concentrations of cisplatin. Viability was assessed at 24 and 72 h. Results show mean +/-SD (n=3, P-values: * ≤ 0.05 ** ≤ 0.01 *** ≤ 0.001). (e): AGS-Gr cells treated with HCQ for 8 h. Protein expression of MAP1LC3B-II and SQSTM1 was detected by immunoblotting. (f) Gastrin induced autophagy is dependent on ULK1: AGS-Gr cells treated with gastrin, BafA1 and ULK1 inhibitor SBI-0206965 (10 μM) for 4 h. Protein expression of MAP1LC3B-II and SQSTM1 was detected by immunoblotting. The immunoblots represent one of three independent experiments. Figure S5: Inhibition of gastrin induced autophagy by Comp C. AGS-Gr cells pretreated with Compound C (10 μM) for 12 h before adding BafA1 and gastrin (4 h). Protein expression of SQSTM1 is shown by immunoblotting. The blot represents one of two independent experiments. (DOCX 2504 kb
