MelJuSo Ub-YFP cells have been previously described [24 (link)]. HOS parental and GFP-LC3B were obtained from Gerald McInerney [58 (link)]. U2OS mRFP-GFP-LC3 were obtained from Jeff MacKeigan [59 (link)]. HeLa TREx FlpIn WT were a kind gift from A. Tighe and S.S. Taylor (University of Manchester, UK) and used to generate the ATG13 and ATG16L1 knockout cell lines, as described below. HEK293 WT cells were obtained from Tamotsu Yoshimori (Osaka University, Japan) and were used to generate ATG16L1 knockout cell lines reconstituted with either eGFP-ATG16L1β or eGFP-ATG16L1[1–249] as described in [40 (link)]. All cells were cultured in DMEM+GlutaMAX (Life Technologies, 31,966–021) supplemented with 10% fetal bovine serum (Life Technologies, 10,270–106) in a humidified incubator at 37°C and 5% CO2. All cell lines were routinely tested for Mycoplasma infection.
To induce autophagy, cells were treated with 250 nM Torin 1 (Tocris, 4247) or with 500 nM KU-0063794 (Sigma-Aldrich, SML0382). Autophagy was blocked using 10 mM 3-methyladenine (3-MA; Sigma-Aldrich, 189,490), 100 nM bafilomycin A1 (BafA1; Enzo Life Sciences, BML-CM110) or 10 µM chloroquine (CQ; Sigma-Aldrich, C6628).
To induce autophagy, cells were treated with 250 nM Torin 1 (Tocris, 4247) or with 500 nM KU-0063794 (Sigma-Aldrich, SML0382). Autophagy was blocked using 10 mM 3-methyladenine (3-MA; Sigma-Aldrich, 189,490), 100 nM bafilomycin A1 (BafA1; Enzo Life Sciences, BML-CM110) or 10 µM chloroquine (CQ; Sigma-Aldrich, C6628).
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