Obatoclax gx15 070

Plasmids expressing GFP-S and hACE2 have been previously described [22 (link)]. The plasmid expressing TMPRSS2 was kindly provided by Prof. Yan Huan (Wuhan University, China). The plasmid with MCL-1 expression was constructed on empty vector pCMV-10 by General Biol Company (Chuzhou, China). Chloroquine (C6628) and N-acetyl-D-glucosamine (GlcN; A3286) were purchased from Sigma-Aldrich (Burlington, MA, USA) and both dissolved in sterile water. Aloxistatin (E64d; HY-100229), bafilomycin A1 (HY-100558), CA074-Me (HY-100350), chlorpromazine (HY-B0407A), MDL 28170 (HY-18236), niclosamide (HY-B0497), ouabain (HY-B0542), and TW-37 (HY-12020) were obtained from MedChemExpress (New Jersey, NJ, USA) and all dissolved in dimethylsulfoxide (DMSO). Obatoclax (GX15-070) was purchased from Selleckchem (Houston, TX, USA) and dissolved in DMSO. LysoTracker Red (Thermo Fisher, L12492) was used as previously described [7 (link)].

U2OS cells were synchronized in mitosis by collection of rounded-up cells after a 2 h treatment with a microtubule poison and then either replated with the drug to maintain mitotic arrest or released in fresh media. Untreated mitotic cells were obtained by washing-off asynchronous cultures. Synchronization at the G1/S boundary by double thymidine block was performed as described [11 (link)]. G1 phase cells were obtained by centrifugal elutriation [19 (link)]. U2OS cells were transfected with Mcl-1 subcloned into pCMV-Flag (MRC Protein Phosphorylation Unit, Dundee) using Superfect transfection reagent (Qiagen). Mcl-1 was depleted in U2OS cells by the siRNA GGACUUUUAGAUUUAGUGA (MWG) using RNAiMax (Invitrogen) as transfection reagent. Cells were treated as indicated with z-VAD-fmk (Enzo Life Sciences), Obatoclax/GX15–070, Navitoclax/ABT-263, NVP-BEZ235 (Selleck Chemicals) or KU55933, NU7441, SB218078 (Tocris).