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Fig. 3 | Molecular Cancer

Fig. 3

From: KCa1.1, a calcium-activated potassium channel subunit alpha 1, is targeted by miR-17-5p and modulates cell migration in malignant pleural mesothelioma

Fig. 3

Molecular and pharmacological inhibition of KCa1.1 inhibits MPM cell line migration. a Transfection with miR-17-5p mimic or KCNMA1-specific siRNA (10 nM) resulted in significantly (* = P < 0.01) decreased KCNMA1 mRNA gene expression in MPM cell lines when compared with controls (Individual P values are included in Additional file 1: Table S5). RT-qPCR carried out as described for Fig. 2. b Immunofluorescent staining of KCa1.1 (Rabbit anti-KCNMA1, 1:500, Sigma) showed significant reduction in expression of KCa1.1 protein in MSTO cells transfected with miR-17-5p mimic or KCNMA1-specific siRNA (final concentration of 10 nM; bar = 400 μm). c Levels of KCNMA1 were measured following AGO2-IP using PCR and were higher in cells transfected with the miR-17 mimic. d Transfecting with miR-17 mimic did inhibit migration of mitotically inactivated H28 cells. Similar results obtained with other MPM cell lines are presented in Additional file 1: Figure S5. e In proliferation assays, the growth of MPM cell lines was inhibited by high concentrations of the KCa1.1 blocker paxilline. In contrast, a sub-lethal dose of paxilline (12.5 μM) inhibited migration (D, last 2 rows) and colony forming ability of MPM cells, plated at low density (f, histograms represent total dye in lysed colonies, as a percentage normalized to control, * P value all < 0.0001). P values for each comparisons are individually presented in the Additional file 1: Table S5. g Levels of cytosolic Ca2+ were estimated by overexpressing the soluble GCaMP5 Ca2+ reporter in MPM cells co-transfected with siRNA. Note, that the fluorescence intensity of the reporter is increased after the KCNMA1 knockdown, * P < 0.05, ANOVA with Holm-Sidak’s multiple comparisons test. h Analysis of the Ca2+ influx in MPM cells overexpressing the membrane-targeted LCK-GCaMP5 Ca2+ reporter and co-transfected with siRNA or miR-17-5p mimic. Calcium influx was induced at 5 s after the start of recording by application of 90 mM K+-containing buffer. Graphs show mean ± SEM fluorescence intensity of the reporter

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