Tunicamycin

To evaluate the effect of ER stress on Notch signaling, human GLCs were preincubated with an ER stress inhibitor, 1 mg/mL tauroursodeoxycholic acid (TUDCA; Tokyo Chemical Industry Co., Tokyo, Japan), for 24 h, and then incubated with ER stress inducers, 2.5 µg/mL tunicamycin (Wako), for 24 h or 1 µM thapsigargin (Sigma-Aldrich), for 6 h. The optimal concentrations of these drugs were chosen based on previous studies using human GLCs [7 (link),9 (link),10 (link)]. To examine the effect of ER stress on expression of genes associated with COC expansion, human GLCs were incubated with 2.5 μg/mL tunicamycin and 50 μM DAPT in DMEM/F-12 containing 10 IU/mL hCG for 6 h. To knockdown activating transcription factor 4 (ATF4), siRNA was obtained from Dharmacon (GE Healthcare) as SMART pools: ON-TARGET plus human ATF4 (L-005125) for knockdown and ON-TARGET plus nontargeting pool (D-001810-10-20) as the negative control. GLCs were transfected with 50 nM siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific) for 24 h. After transfection, the medium was removed, and GLCs were incubated with 2.5 μg/mL tunicamycin for 24 h.

Prior to exposure to mitochondrial-damaging agents and/or antioxidants, synchronized young adult worms were washed from NGM plates seeded with OP50, and then resuspended in S Basal supplemented with one of the following: 50 μM antimycin A (Sigma), 50 μM rotenone (Sigma), 3 mM TTFA (Sigma), 10 mM sodium azide (Sigma), 20 μM CCCP (Sigma), 10% ethanol (Fisher), 1 mM phenanthroline (Sigma), 7 mM sodium selenite (Alfa Aesar), 1 mM H₂O₂ (Sigma), 60 μM tunicamycin (Thermo Fisher), 12.5 μM bortezomib (Thermo Fisher), 50 μM juglone (Sigma), 5 or 2.5 mM NAC (Acros Organics), 25 mM ascorbate (TCI), 5 mM TEMPOL (Cayman Chemical), and 10 μM mitoquinol (Cayman Chemical) in the presence of OP50. When specified, treatments were combined (e.g., rotenone/NAC, rotenone/NAC/ascorbate, etc.). Worms were imaged every 2 h for 20 h. Three biological replicates per experiment were performed, with ~ 400 worms per replicate.

The cells (S, R, and T; inoculums: 1 × 10⁶ cells) were cultured in 4 mL RPMI 1640 media with L-glutamine (1 μg/mL), 8% fetal bovine serum, and 1 μg/mL gentamycin (all purchased from Gibco, Langley, OK, USA) in a humidified atmosphere with 5% CO2 at 37 °C for 48 h in the absence or presence of tunicamycin (0.1 μM). This procedure was termed passaging and was repeated three times. The number of viable cells after each passage was counted using a CASY Model TT Cell Counter (Roche Applied Sciences, Madison, WI, USA). R cells were cultured for two passages without vincristine (VCR) prior to the experiments.

The following three L1210 cell variants were used in this study: (i) S-drug-sensitive parental cells obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) ACC-123; (ii) R—P-gp-positive drug-resistant cells that overexpress P-gp after selection with vincristine [44 (link)] obtained from Gedeon Richter Co., (Budapest, Hungary); and (iii) T—P-gp-positive drug-resistant cells that overexpress P-gp following stable transfection with the P-gp gene [15 (link)], using the Addgene plasmid 10957 (pHaMDRwt), a retrovirus encoding the full-length P-gp cDNA [45 (link)]. The cells (S, R and T; inoculums 1 × 10⁶ cells) were cultured in 4 cm³ RPMI 1640 media with l-glutamine (1 mg cm⁻³), 4% fetal bovine serum and 1 μg cm⁻³ gentamycin (all purchased from Gibco, Langley, OK, USA) in a humidified atmosphere with 5% CO₂ and air at 37 °C for 48 h in the absence or presence of either tunicamycin (0.1 μmol·dm⁻³) or GalNAc-α-O-benzyl (0.1 mmol·dm⁻³). This procedure was termed as passage and was repeated three times. Numbers of viable cells after each passage were counted using a CASY Model TT Cell Counter (Roche Applied Sciences, Madison, WI, USA). R cells were cultured for two passages without VCR prior to the experiments.