Concanamycin A

Cells were grown exponentially for 15 hours to a maximum density of 5 × 10⁶ cells/ml prior to the initiation of all experiments. Because nutrients and growth phase affected vacuolar and mitochondrial function, extended log-phase growth was necessary to ensure vacuolar and mitochondrial uniformity across the cell population prior to the initiation of all experiments. Cells were cultured in YEPD (1% yeast extract, 2% peptone, 2% glucose) unless otherwise indicated. For CR experiments, cells were cultured in YEP plus the indicated carbon source. Yeast Complete (YC) medium used in the high copy suppressor screen was previously described^{37 (link)}. Concanamycin A (Sigma) was added to cultures at a final concentration of 250 or 500 nM as indicated in figure legends.

4-hour chromium release assays (CRA) and luciferase-based cytotoxicity assays (LCA) were performed as previously described (15 (link), 16 (link)). When specified, tumor cells were peptide loaded in neural stem cell media at a final concentration of 10 μg/mL peptide for 2 h at 37°C. For concanamycin (CMA) inhibition, CTL were resuspended at 10⁶ cells/mL and incubated for 2 hr at 37° C in the absence or presence of CMA (Calbiochem® EMD Chemicals, Inc., Gibbstown, NJ) prior to co-culture with tumor cells at a 10:1 effector:target (E:T) ratio.
CD107a degranulation assays were performed as described (17 (link)). For the flow based cytotoxicity assay, tumor cells were stained with 0.1 μM CFSE as per manufacturer's instructions (CellTrace™ CFSE Cell Proliferation Kit, Molecular Probes, Eugene, OR), peptide loaded, and then stained with APC-conjugated anti-CD133/1 and anti-CD133/2 (Miltenyi). The plates were incubated for 4 hr at 37°C, harvested and re-suspended with calibrite beads (1 drop/10 mL; BD Biosciences) and PI (0.5 μg/mL). Samples were run on the Cyan flow cytometer (Dako, Carpinteria, CA) and events collected were limited to a constant number of beads. Cytotoxicity was calculated based on the number of viable (CSFE⁺ PI⁻) tumor cells that were present at various E:T ratios and normalized to that obtained with control a CD19-specific CTL line.
Cytokine production was measured by co-culturing T cells with tumor at a 10:1 E:T ratio. After 20-24 hr incubation, supernatants were harvested and assayed using Luminex multiplex bead technology (Upstate, Waltham, MA), and output data were analyzed via the Bio-Plex Manager 4.0 (BioRad, Hercules, CA).