Fig 2: Role of H+, K+-ATPase in PDAC cell proliferation, cell viability and cell cycle. (A) Effect of PPIs and P-CAB on cell proliferation in HPDE (100 µM SCH-28080: ** p = 0.0013; 1 µM Ome+10 µM SCH-28080:* p = 0.0114; 10 µM Ome+100 µM SCH-28080: *** p = 0.0002), BxPC-3 (10 µM Ome: *P= 0.0485; 10 µM SCH-28080: ** p = 0.003; 100 µM SCH-28080: ** p = 0.001; 1 µM Ome+10 µM SCH-28080: ** p = 0.0084; 10 µM Ome+100 µM SCH-28080: **** p < 0.0001) and Capan-1 (1 µM Ome: *** p = 0.0006; 10 µM Ome: *** p = 0.0002; 10 µM SCH-28080: ** p = 0.0083; 100 µM SCH-28080: * p = 0.0233; 1 µM Ome+10 µM SCH-28080: ** p = 0.0042; 10 µM Ome+100 µM SCH-28080: *** p = 0.0006) cell lines; one-sample t-tests. Cells were incubated for 24 h with two different concentrations of omeprazole and SCH-28080, individually and together. (B) The effect of various concentrations of pantoprazole on proliferation of PANC-1 (50 µM:** p = 0.0042; 100 µM:*** p = 0.0008) and MIA PaCa-2 cells (100 µM: * p = 0.0168), one-sample t-tests and p values adjusted for multiple comparisons as more than two different conditions were tested against controls. (C) Dose-response curve for pantoprazole on PANC-1 cell proliferation. (D) Effect of pantoprazole treatment on PANC-1 spheroid sizes (* p = 0.0273 (one-sample t-test)). (E) Effect of three different siRNAs targeted to HKa1 on PANC-1 cell proliferation (* p = 0.012) with respective western blot showing the effectiveness of the siRNAs on HKa1 protein expression. (F) Effect of various concentrations of pantoprazole on LDH (lactate dehydrogenase) release in PANC-1 cells. (50 µM: *** p = 0.0008; 100 µM: * p = 0.012 (adjusted P values, one-sample t-test) (G) Effect of pantoprazole on the different phases of the cell cycle in PANC-1 cells: G0/G1 (** p =0.0087; ** p =0.0013); S (NS); G2/M (* p =0.0113); paired t-test). Peak count analysis is shown in the right panel. Data are shown as means ± s.e.m. from the indicated number of independent experiments.

Fig 3: Immunolocalization of H+/K+-ATPases in sections of human pancreas. A: The gastric H+/K+-ATPase a subunit (HKa1) was labeled with Calbiochem 119101 (polyclonal, against HKa1C-terminal) (image 1 and 2) and Calbiochem 119102 (polyclonal, against HKa1 N-terminal) and Alexa 488 (green); B: The gastric H+/K+-ATPase ß subunit (HKß) was stained with Sigma A-274 (2G11, anti HKß, monoclonal) and Alexa 488 (green). C: The non-gastric H+/K+-ATPase a subunit (HKa2) was stained with non-gastric HKa2 antibody (C384-M79) and Alexa 488 (green). D: Example of a control image without primary antibodies. DAPI was used to stain the nucleus (blue). All bars are 25 µm, and images are from 3 independent experiments and show localization in ducts of various sizes.

Fig 4: Expression of H+, K+-ATPase subunits HKa1, HKa2 and HKß in human pancreatic duct cell lines. (A) Quantitative RT-PCR showing the mRNA expression of the three subunits relative to HPDE. Data are shown as means ± s.e.m. from indicated number of independent experiments. One-sample t-test with Holm-Bonferroni correction was used for statistic: ATP4A: * p = 0.0145, ATP12A: **** p < 0.0001; ** p = 0.0012 and * p = 0.0116 and 0.0151. Unadjusted p value for ATP4B in PANC-1 and MIA PaCa2 were 0.0473 and 0.0280. (B) Representative Western blot for HKa1 subunit (Abcam EPR12251), HKa2 subunit (Sigma, HPA039526) and HKß subunit (Sigma A274) on the non-cancer cell line HPDE, and PDAC cell lines AsPC-1, BxPC-3, Capan-1, MIA PaCa-2 and PANC-1, as well as control tissues (mouse stomach and colon). Loading control was ß-actin. (C) Immunolocalization of HKa1 (Calbiochem 119101, 1:100), HKa2 (C384-M79, 1:300) and HKß (Sigma A274, 1:100) is reported in green. DAPI (blue) was used to stain the nuclei. Scale bars indicate 25 µm.

Fig 5: Expression and function of H+, K+-ATPase in human pancreatic stellate cells PaSC. (A) Representative gel of ATP4A (HKa1), ATP12A (HKa2) and ATP4B (HKß) mRNA expression. (B) Representative western blot of HKa1 and HKß protein expression. Loading controls were ß-actin and vinculin. (C) Immunolocalization of HKa1 (Calbiochem 119101, 1:100), HKa2 (C384-M79, 1:300) and HKß (Sigma A274, 1:100) is shown in green. DAPI (blue) was used to stain the nuclei. Scale bars indicate 25 µm. (D) Effect of pantoprazole on PaSC proliferation with 100 µM pantoprazole (* p = 0.0153) and Aphidicolin (5 µM) was used as a negative control (**** p < 0.0001), one sample t-test corrected for multiple comparison with Holm-Bonferroni method. (E) Effect of pantoprazole on collagen production induced by TGF-ß1 (1 ng/ml) alone (*** p = 0.0002), or combined with pantoprazole 50 µM (** p = 0.0017) and 100 µM (* p = 0.0153), paired t-test. Data are shown as means ± s.e.m. from the indicated number of independent experiments.