Additional file 1: Figure S1a. of Gastrin activates autophagy and increases migration and survival of gastric adenocarcinoma cells

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