Concanamycin a

Quinacrine (Sigma) staining was performed as previously described^{41 (link)} (Hughes and Gottschling). Briefly, cells were washed once in YEPD + 100 mM HEPES, pH 7.6 and resuspended in 100 μl of the same buffered media containing 200 μM Quinacrine. Cells were incubated for 10 min at 30 °C and then 5 min on ice, followed by pelleting and washing twice with ice cold 100 mM HEPES, pH 7.6 + 2% glucose. Cells were resuspended in 100 mM HEPES, pH 7.6 + 2% glucose for imaging. Quinacrine staining was performed for cells in several conditions: (i) exponentially growing cells from 2% glucose, (ii) cells after 90 min of starvation in 0.2% glucose, (iii) cells after 90 min of starvation in 0.02% glucose, (iv) cells after a 20-min recovery in 2% glucose following starvation in 0.2% glucose, and (v) cells after a 20-min recovery in 2% glucose following starvation in 0.02% glucose. Prior to imaging, cells were kept on ice and all images were obtained within 30 min of staining. Supplementary Fig. 17b presents an example of quicacrine stained (acidified) vacuole, and only such vacuoles were scored as positive for Quinacrine staining, i.e., acidified. Concanamycin A (Sigma) was added to cultures at a final concentration of 500 nM to achieve the inhibition of vacuolar acidification.

As previously described (Hughes and Gottschling, 2012 (link)), cells were grown exponentially for 15 hr to a max density of 5 x 10⁶ cells/ml before the start of all aging and MDC assays. This period of overnight log-phase growth was carried out to ensure vacuolar and mitochondrial uniformity across the cell population. Cells were cultured in YEPD (1% yeast extract, 2% peptone, 2% glucose) for all experiments. Yeast Complete (YC) medium used during construction of the Tom70-mCherry GFP strain collection was previously described (Tong and Boone, 2006 (link); van Leeuwen and Gottschling, 2002 (link)). Concanamycin A (Sigma-Aldrich, St. Louis, MO) was added to cultures at a final concentration of 500 nM as indicated in figure legends. In the RITE tag experiments, cycloheximide (Sigma-Aldrich) was added at a final concentration of 50 μg/ml, and β-estradiol at 1 μM. In Figure 3, FCCP (Sigma-Aldrich), Antimycin A (Sigma-Aldrich), and hydrogen peroxide (Sigma-Aldrich) were added at to cultures at final concentrations of 10 μM, 20 μg/ml, and 3 mM, respectively.

Bafilomycin A1 (BM), concanamycin A (CCM), chloroquine (CQ), 3-methyladenine (3-MA), 5-fluorouracil (5-FU), and 2-phenylethynesulfonamide (PES) were purchased from Sigma-Aldrich (St. Louis, MO, USA). siRNAs were obtained from Bioneer (Deajeon, Korea). The following antibodies were used: Bcl-2 (ab32124, Abcam, Cambrige, MA, USA), Bcl-xl (2764, Cell Signaling Technology), Bak1 (3814, Cell Signaling Technology), ATP6V0C (ab104374, Abcam), LC3B (2775, Cell Signaling Technology, Beverly, MA, USA), PARP-1 (9542, Cell Signaling Technology), PIK3CA (4249, Cell Signaling Technology), HA (2367, Cell Signaling Technology), Myc (sc-789, Santa Cruz Biotechnology) and GAPDH (LF-PA0212, AbFrontier Co., Ltd, Seoul, Korea).

In vitro cytotoxiocity assay was performed using CFSE-based labeling6 (link). SK37 or peptide-pulsed SK29 were labeled with 0.5 μM CFSE, whereas peptide-unpulsed SK29 was labeled with 5 μM CFSE. In some experiments, cancer cells were treated with 20 μg/ml Fas-ligand antibodies (eBiosciences) for 1 hour before addition to T cells. Fas-ligand antibody was also present during co-culture. 2 U/ml of recombinant IL-2, combination of 10 μg/ml anti-CD25 (IL-2Rα) antibody (BD Bioscience) and anti-IL-2 antibody (eBioscience), 0.1 nM concanamycin A (Sigma-Aldrich) or 100 μM Z-AAD-CMK (Enzo Biochem, Inc.) were added at the initiation of T cell culture. Cells were incubated in 5 mL round-bottom tubes for 14–16 hours. Cancer cells were harvested by treatment with trypsin/EDTA and analyzed for CFSE staining levels by FACSCalibur flow cytometer (BD Biosciences) to enumerate the percentages of SK37 and SK29. Acquisition data were analyzed by FCS Express Version 3 software (De Novo Software) or FlowJo software. Cytotoxicity was calculated using the following formula: % cytotoxicity = 100 × {1−(%SK29/%SK37)_{without T cells}/(%SK29/%SK37)_{with T cells}}.

As lysosomal-inhibiting agents, we used the following: (a) 10 μM Leupeptin inhibitor of cysteinyl and serine proteases) and Pepstatin A (aspartyl proteases)(Peptide Institute, Japan) for 24 h; (b) 10 nM Bafilomycin A (Bafil), a specific inhibitor of the lysosomal vacuolar type H⁺-ATPase (V-ATPase) that blocks lysosomal degradation^{28 (link)} (Sigma B1793) for 6 h; (c) 50 nM Concanamycin A (ConA), a second potent V-ATPase inhibitor^{29 (link)} (Sigma 27689) for 2 h. For mitochondria depletion via induced mitochondrial membrane depolarization, cells were treated with 20 μM CCCP^{15 (link)} (Sigma C2759) for 6 h or with a combination of 1 μM antimycin A and 1 μM oligomycin (AA/OA) inhibiting the electron transport chain complex III and of ATP synthase, respectively^{28 (link)} (Sigma A8674/O4876) for 1, 3, 6 and 24 h. Cells were treated with 0.1 μM AZD8055 (AZD), a recently introduced ATP-competitive inhibitor directly targeting the mTOR catalytic site, blocking both mTORC1 and mTORC2^{30 (link)} (Selleckchem) for 2, 4 and 8 h as reported in figure legends.

The polyclonal antibody directed against the N-terminal of NBCn1 was kindly provided by Jeppe Prætorius, Aarhus University, Denmark. Antibodies against ZO-1 (sc-10804), and GFP (sc-8334) were from Santa Cruz Biotechnology, Texas, USA. Antibodies directed against RACK1 (#610177 and #A2560) and E-cadherin (#610181) were from BD Transduction Laboratories, San Jose, USA and ABclonal, Woburn, USA, antibody against Cyclin B1 (#12231) was from Cell Signaling Technology, Danvers, USA, and antibodies against biotin (#B3640) and ß-actin (#A5441) and FLAG M2 (#F1804) were from Sigma-Aldrich, St. Louis, USA. Cycloheximide (#C7698) and Concanamycin A (#C9705), both from Sigma-Aldrich, were dissolved at 5 mg/mL in ddH₂O and 10 µM in DMSO, respectively.