Fig 7. MitoKCa3.1 regulates mitochondrial respiration in Mia PaCa-2 cells.
(A) Western Blot analyses of Mia PaCa-2, BxPC-3, Panc-1 and Capan-1 cells; mitochondrial fraction was prepared from equal cell number of each cell line, supernatant from mitochondrial fraction preparation was taken as the cytosolic fraction. Fractions were compared among each other and to whole cell protein lysates. Antibodies recognizing mitochondrial complex V, III, II, IV were used as mitochondrial controls and tubulin as the cytosolic control. Arrow indicates the KCNN4 protein band. (B) OCR measurement of permeabilized in Mannitol and Sucrose buffer (MAS) by 25 μg/ml saponin (SAP) Mia PaCa-2 cells transfected with individual siRNAs against KCNN4, 30 000 cells/well were re-seeded the night before the assay to avoid cell number-dependent changes, n = 4 for each condition, p<0.001. (C) OCR measurement of permeabilized in Mannitol and Sucrose buffer (MAS) by 25 μg/ml saponin (SAP) Mia PaCa-2 cells treated with 10 μM KCa3.1 inhibitor rac-16, n = 32, p<0.01; (D) Mitochondrial ATP production in Mia PaCa-2 cells upon treatment with varying concentrations of the KCa3.1 inhibitor rac-16 or DMSO, 1 μM Oligomycin (Oligo) served as a positive control, n = 6, p<0.0001. Bonferroni ANOVA test was performed to compare Non-Targeting siRNA vs siRNA targeting KCNN4 mRNA or DMSO-vs rac-16 treatment; ns, not significant, p>0.05, *, p-value is 0.01 to 0.05; **, p-value < 0.01.