Immunofluorescence (IF) detection was performed as previously described [58 (link)]. Briefly, UMUC3 and J82 cell transfectants were cultured on cover slides in 10% FBS-supplemented DMEM or MEM in each well of 24-well plates. After synchronization, cells were cultured in complete medium for 12 h, followed by 0.1% FBS medium for another 12 h. The cells were fixed with 4% paraformaldehyde (Sigma Aldrich Corporation, 158127) in PBS (Gibco, 8117296) at room temperature (RT) for 15 min, followed by permeabilization in 0.25% Triton X-100 (Beyotime, ST795) at RT for 15 min and staining with LC3A/B (1:100; Cell Signaling Technology, D3U4 C) for 12 h at 4°C. The slides were washed three times with PBS and stained with 0.1 mg/mL DAPI (Sigma Aldrich Corporation, 9542) and CY3 (1:100; BOSTER, BA1032) for 60 min at RT. The slides were washed three times with PBS and once with ddH2O, and then fixed in glycerin. Cell images were captured using an inverted Nikon fluorescence microscope (A1 R). For the quantification of autophagic cells, puncta were determined in 20 random cells per slide.