Lc3 1

In order to create protein lysates from MRC-5, A549, Calu-3, H1975, and H2228, protease and phosphatase inhibitors were added to radioimmunoprecipitation assay (RIPA) lysis solution (Thermo Fisher Scientific). The bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) was used to measure the amounts of proteins. After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for electrophoresis, polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA) were transferred. Immunoblotting was carried out using primary antibodies against CDC25C, Bax, Bcl-2, LC3 I, LC3 II, p62, cyclin B, CDK1-Y15, CDK1, c-PARP, and GAPDH (all at 1:1,000; Abcam, Cambridge, MA, USA). Post-washing thrice with tris-buffered saline with Tween 20 (TBST), membranes were processed for band visualization using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific). Using the ChemiDoc system from Bio-Rad (Hercules, CA, USA), the intensities of the protein bands were recorded and subsequently analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Proteins were obtained from tissues and cells by RIPA Buffer (Beyotime, Shanghai, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime) was applied to separate protein. Subsequently, proteins were transferred to 0.22 μm polyvinylidene difluoride (PVDF, Beyotime) membranes. After blocked in 5% non-fat milk for 2 h, the membranes were incubated overnight at 4^°C with the primary antibodies against multi-drug resistance gene1 (MDR1, 1:200, Thermo Fisher Scientific), Light chain3-I (LC3-I, 1:2000, Abcam, Cambridge, MA, USA), LC3-II (1:1000, Abcam), P62 (2 μg/mL, Abcam), CDK8 (1:2000, Abcam) or GAPDH (1:3000, Abcam). The membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit second antibody (1:4000, Abcam) for 1 h. The bands were observed by ECL reagent (R&S, Shanghai, China) and analyzed using ImageJ analysis software.

Cells were lysed for 30 min on ice in Radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor (Roche). BCA kit (Beyotime Institute of Biotechnology, Shanghai, China) was employed to determine protein concentration. The total protein sample was loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer before being transferred to polyvinylidene difluoride (PVDF) membranes, which were sealed with 5% skim milk at 37 °C for 120 min. The membranes were then incubated with the following primary antibodies: Bcl-2, Bcl-2-associated X protein (Bax), Cleaved caspase-3, ATF4, GRP78, CHOP, Beclin4, p62, LC3I, LC3II, p-P13K, p-AKT, p-mTOR and β-actin (all purchased from Abcam, Cambridge, MA, USA) at 4 °C overnight. After that, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The signals were visualized with the enhanced Horseradish Peroxidase (Pierce, Rockford, IL, USA). An automatic digital gel image analysis system Bio-Rad CFX-96 (Bio-Rad, CA, USA) was used to determine and analyze the density of the bands.