Atg4b

The AGS, MKN45, HGC27, SNU1, KATOIII, Hela and HEK293T cells were obtained from the American Type Culture Collection (ATCC). Human GCa cell lines MGC803 were purchased from China Academia Sinica (Shanghai, PR China). The GCa MGC803, AGS, MKN45, HGC27, SNU1, KATOIII cells were cultured in RPMI-1640 medium and Hela cells, HEK293T embryonic kidney cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO₂. Antibodies against the following proteins were used with source and dilution ratios indicated: ATG4B (Cell signaling, #13507, 1:1000 and Proteintech, # Cat No. 15131-1-AP, 1:1000); P62 (Sigma, #P0067, 1:1000); LC3 (Sigma, #ABC929, 1:1000; immunofluorescence 1:100); LAMP1(CST, #15665S, 1:100); Snail (CST, #4719, 1:1000); N-cadherin (CST, #4061, 1:1000); C-caspase7 (CST, #12827, 1:1000); PARP-1 (CST, #9542, 1:1000); GAPDH (CST, #2118, 1:1000); Anti-rabbit IgG Fab2 (CST, #4412s, 1:500); Anti-mouse IgG Fab2 (CST, #4409s, 1:500). AzalomycinF4a was isolated from Streptomyces solisilvae HNM30702 and verified by the NMR and HRESIMS data [34 (link)]. Rapamycin (MCE, #HY-10219); BafilomycinA1 (MCE, # HY-100558); Tioconazole (MCE, #HY-1303191 CS-2360); Acridine Orange (Sigma, #MKCD9806); FMK9a (MCE, HY-100522); DAPI (Beyotime, ON.C1005).

Primary antibodies and dilutions used for Western blotting were as follows: ATG3 C-ter (1:1,000, Abcam, ab108282), ATG3 N-ter (1:1,000, Abcam, ab108251), ATG4B (1:1,000, Cell Signaling Technology, no. 5299), β-actin (1:4,000, Sigma, A1978), FLAG biotin-conjugated (1:1,000, Sigma, F9291), GFP (1:2,000, Clontech, no. 632381), LC3A/B (1:500, Cell Signaling Technology, no. 12741), LC3B (1:1,000, Sigma, L7543), SQSTM1 (1:1,000, Sigma, P0057), vinculin (1:1,000, Abcam, ab129002).
Secondary antibodies for Western blotting with chemiluminescence-based detection were as follows: goat anti-rabbit/anti-mouse IgG, HRP-linked (1:5,000, Cell Signaling Technology, no. 7074/no. 7076). For fluorescence-based detection, the following were used: IRDye 800CW goat anti-rabbit/anti-mouse (1:15,000 with 0.02% SDS, LI-COR no. 926-32211/926-32212); IRDye 680LT goat anti-rabbit/anti-mouse (1:25,000 with 0.02% SDS, LI-COR no. 926-68021/926-68020).
Torin1 was purchased from Merck-Millipore (no. 475991), and bafilomycin A₁ from Streptomyces griseus was purchased from Sigma (B1793); both were dissolved in DMSO. Recombinant GST–ATG4B WT and C74S were produced and purified as described previously (65 (link)). Earle's balanced salt solution (EBSS) containing calcium and magnesium was purchased from Thermo Fisher Scientific (no. 24010-043).

Monoclonal antibody against MAP1S (Cat# AG10006) was a gift from Precision Antibody^™, A&G Pharmaceutical, Inc., Columbia, Maryland. Primary antibodies against GFP (SC‐8334), β‐actin (SC‐47778), P27 (SC‐776), EEA1 (SC‐33585), Beclin 1 (SC‐11427), phosphorylated Bcl‐2 (p‐Bcl2, SC‐377576), phosphorylated p27 (p‐p27, SC‐130603), and ATG5 (SC‐33210) were purchased from Santa Cruz Biotechnology, Inc., Dallas, Texas, USA. Antibody against human LC3 (NB 100‐2331) was from Novus Biologicals. Antibodies against α‐SMA (ab‐5694), fibronectin (ab2413), LAMP2 (ab18528), and TGF‐β (ab66043) were from abcam, Cambridge, Massachusetts, USA. Antibodies against γ‐H2AX (9718 S), Bcl‐2 (#2870), PI3KCIII (#4263), H2AX (#2595), and ATG4B (#5299) were from Cell Signaling Technology, Danvers, Massachusetts, USA. Antibody against p62 (BML‐PW9860) was from Enzo Life Sciences International Plymouth Meeting, Pennsylvania, USA. Horseradish peroxidase‐conjugated secondary antibodies against mouse (#172‐1011) and rabbit (#172‐1019) were from Bio‐Rad, Hercules, California, USA. Rhodamine Red‐X goat anti‐mouse IgG (R6393) and FITC rabbit anti‐mouse IgG (A21202) were from Invitrogen, Carlsbad, California, USA. RFP‐LC3 was a gift from Dr. Mizushima (Mizushima et al., 2004). Bafilomycin A1, Sirius Red (Direct Red 80, 365548) and Hydroxyproline Assay Kit (MAK008‐1KT) were from Sigma, St. Louis, Missouri, USA.