Minitab

The expression of p62, a classical marker of autophagy, in PANC-1 cells exposed to ¹³¹I-FAP-2286, was measured by western blotting. To assess the influence of CAFs on the autophagy of PANC-1 induced by ¹³¹I-FAP-2286, the cell co-culture experiment was performed in a 6-well plate with 3 μm pore size transwell inserts, where PANC-1 cells and CAFs were seeded into the bottom wells and the upper inserts, respectively. After a 24-h culture with ¹³¹I-FAP-2286, p62 expression in PANC-1 was detected. After receiving the corresponding treatments, the cells were lysed using RIPA lysis buffer in the presence of protease and phosphatase inhibitors (Minitab, Roche). The lysates were sonicated on ice and then centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatants were collected for the protein concentration detection, and then were exposed to 12% SDS–polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes, followed by being blocked with Odyssey blocking buffer (LI-COR) in PBS containing 1% Tween-20. Primary antibodies of p62 and β-actin were incubated in blocking buffer at 4 °C overnight, and then were detected using HRP-conjugated secondary antibody. The photos of the target protein bands were captured using LI-COR Odyssey protein imaging system and quantified by Image J software.