Sqstm1

Cells were seeded with a density of 200.000 cells/well in a 24-wells format and incubated for 48 h under a 37 ${}^{\circ }$ C and humidified 5% CO ${}_2$ atmosphere environment. After 48h of incubation the cells were exposed to either DMSO, diethylmaleate or diclofenac for 3, 8 or 24h. After exposure cells were lysed and RNA was isolated using the RNA isolation kit (Macherey Nagel GmbH, Düren, Germany). RNA concentrations were measured by nanodrop and cDNA was constructed using the REVERTAID H MINUS cDNA kit (Thermo Scientific, Leiden, The Netherlands). Kicqstart primers for TBP, KEAP1, NFE2L2, SRXN1 and SQSTM1 (Sigma Aldrich, Zwijndrecht, The Netherlands) were used to detect mRNA levels using POWRUP SYBR master mix (Thermo Scientific, Leiden, The Netherlands). Cycle time values were obtained from a QuandStudio 6 Flex qPCR machine (Thermo Scientific, Leiden, The Netherlands). Cycle time values exceeding the threshold were normalized to the housekeeping gene TBP and used as a quantification of the qPCR.

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).