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. 2016 Dec 2;7:13297. doi: 10.1038/ncomms13297

Figure 1. L-type calcium channel blockers (CCBs) inhibit filopodia formation and L-type calcium channels are functional in cancer cells.

Figure 1

(a) MDA-MB-231 cells transiently expressing MYO10-GFP and adhering to fibronectin (FN) were treated with various compounds (10 μM) for 1 h, fixed, stained for actin and imaged on a TIRF microscope (scale bar, 20 μm). The number of MYO10-positive filopodia was counted for each cell and displayed as a box plot (three biological repeats, n>100 cells, ***P value<8.3 × 10−17). (b) Relative expression of the four genes encoding the L-type calcium channel α1 subunit across 676 commonly used cancer cell lines19. Gene expression read counts are displayed. The value 7.99 corresponds to non-detected (ND). Selected cell lines are annotated. (c) MDA-MB-231 cells transiently expressing the calcium probe (pGP-CMV-GCaMP6s) and adhering to FN were treated with an L-type calcium channel activator (BAY K8644; 1 μM) while being imaged on a TIRF microscope (63 × objective). The relative increase in the intracellular intensity of the calcium probe was measured at 1 and 10 min post stimulation. Cell boundaries are indicated by dotted lines (three biological repeats, n=74 cells; scale bar, 20 μm). (d) MDA-MB-231 cells transiently expressing the calcium probe (pGP-CMV-GCaMP6s) and MYO10-mCherry were seeded on FN and treated with an L-type calcium channel activator (BAY K8644; 1 μM) while being imaged on a TIRF microscope (100 × objective; scale bar, 10 μm). The inset shows a representative MYO10-positive filopodia tip delineated by a dotted line. ROI: region of interest. (e) MDA-MB-231 cells transiently expressing the calcium probe (pGP-CMV-GCaMP6s) and adhering to FN were treated with an L-type calcium channel activator (BAY K8644; 1 μM) in combination with DMSO or amlodipine besylate (1 μM). The relative increase in the intracellular intensity of the calcium probe was measured at 1 min (three biological repeats, n>21 cells; ***P value<1.39 × 10−4). P values were calculated using Student's t-test (unpaired, two-tailed, unequal variance).