Fig. 1

Short-term response and long-term adaptation of PDAC tumor cells to acidic pHe stress. a Scheme of PDAC cells exposed to various periods of extracellular acidification. Five cell groups were established including the control group (Ctrl) cultured in RPMI-1640 medium at pHe 7.4; the buffered group (Buff) in which cancerous cells were cultivated in RPMI-1640 medium supplemented with HEPES/PIPES buffer at pHe 7.4; and the three acid-treated groups with tumor cells incubated in buffered medium at mild pHe 6.7 for a short period of 2-3 weeks (S.A., short-term acidification), a medium period of 4-5 months (M.A., medium-term acidification), or a long period of 10-12 months (L.A., long-term acidification). b The time effects of acidic pHe stress on the proliferation of SUIT-2 cells were evaluated with MTT assay; data are shown as the mean ± SD (n = 3) and as fold-change relative to day 0. c-d The time effects of extracellular acidification on cell viability and cell cycle progression. Asterisks represent significant differences between the indicated groups versus the Buff group (means ± SD, n = 3). e Western blots of cell cycle-related proteins under various control and acidic pHe stress conditions. f The time effects of extracellular acidification on cell phenotype and autophagy. The bottom panels display enlargement of the yellow dotted-boxed area from the respective top panels. Scale bar: 50 μm. g Accumulation and clearance of autophagic vacuoles and autolysosomes (autophagosome-lysosome fusion) in response to different periods of acidic pHe exposure. Scale bar: 50 μm. h-i Representative blots of autophagy-related markers and pro-survival proteins. j-m Seahorse assays for SUIT-2 cells under various control and acidic pHe conditions with oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) displayed as mean ± SD (n = 4). FCCP: carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone; NS: not significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001