Ptflc3 plasmid

To perform this assay, 1 × 10⁵ 6606PDA cells per well or 4 × 10⁵ MIA PaCa-2 cells per well were plated in a glass-bottom dish (NEST, Wuxi, China, code 801001). On the following day, the cells were transfected with the ptfLC3 plasmid (Addgene, Cambridge, UK, code 21074), which was a kind gift from Tamotsu Yoshimori [17] (link), and Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, USA, code L3000001). After 24 h, the cells were treated with medium containing DMSO (Sham) or LW6 (80 µM for MIA PaCa-2, 160 µM for 6606PDA) for another 12 h and then were fixed with 4% formalin. The nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI). The images were acquired by a confocal microscope, Zeiss LSM 780 (Zeiss, Oberkochen, Germany), using the 60× oil objective. Only cells that were transfected with the ptfLC3 plasmid were evaluated. To evaluate the ratio of autophagosomes/cell in each field, the following formula was used: autophagosomes/cell = the number of yellow dots/the number of nuclei. Similarly, the ratio of autolysosomes/cell was defined as the number of red dots divided by the number of nuclei in each field. For each experiment, five fields per treatment were randomly acquired and evaluated.