Staurosporine sts

For drug treatment experiments, stock solutions of paclitaxel (Sigma Aldrich), 5-FU (Sigma Aldrich), HA14-1 (Cayman Chemical), and staurosporine (STS) (Cell Signaling Technology) were diluted in DMSO, whereas water was used to dilute a stock solution of carboplatin (Sigma-Aldrich). For the treatment of cells overexpressing TMPRSS13, DLD-1 cells were subjected to 48-h treatments with paclitaxel (10 µM), carboplatin (50 µM), or 5-FU (100 µM) starting 24 h after transient transfection with plasmid vectors. Twenty-four hours after transient transfection with an expression vector, DLD-1 and HCT116 cells were subjected to treatment with HA14-1 or STS. The duration of HA14-1 treatment for DLD-1 cells was 1.5 h at a concentration of 10 µM and a duration of 4 h for STS treatment at a concentration of 1 µM. In HCT116 cells, HA14-1 treatment lasted 1 h at 60 µM. All drug treatment conditions were concluded by lysate collection. For drug treatment of TMPRSS13-silenced cells, siRNA-treated DLD-1 cells were subjected to a 48-h treatment of paclitaxel at a final concentration of 10 µM (48 h post-transfection) and lysates were collected four days post-transfection. Four days post-transfection, siRNA-treated DLD-1 and HCT116 cells were subjected to a 1-h treatment of HA14-1 at final concentrations of 30 and 60 µM.

Antibodies against Parkin (4211), cleaved caspase 3 (9664), poly(ADP-ribose) polymerase (PARP) (9542), c-Jun (9165), and voltage-dependent anion channel (VDAC) (4866), phospho-SAPK/JNK (Thr183/Tyr185) (9251), horseradish peroxidase (HRP)-linked anti-mouse IgG (7076) were purchased from Cell Signaling Technology (Danvers, MA, United States). Antibodies against p62 (P0067, Sigma-Aldrich, Saint Louis, MO, United States), LC3B (100-2220, Novus Biologicals, City of Centennial, CO, United States), HRP-conjugated β-actin (47778, Santa Cruz Biotechnology, Dallas, TX, United States) and goat anti-rabbit IgG (H+L) secondary antibody (31460, Thermo Fisher Scientific, Carlsbad, CA, United States), PTEN-induced putative kinase 1 (PINK1) (BC100-494, Novus Biologicals) and ubiquitin phosphorylated at S65 residue (p-Ub-S65) (ABS1513-I, EMD Millipore, Burlington, MA, United States) were purchased at the indicated companies. Bafilomycin A1 (Baf.A1, Sigma-Aldrich), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma-Aldrich), staurosporine (STS, Cell Signaling Technology), and necrostatin-1 (Invitrogen, Carlsbad, CA, United States) were purchased from the indicated companies.