Opening large-conductance potassium channels selectively induced cell death of triple-negative breast cancer

Figures

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  Fig. 1. KCNMA1 gene expression in five molecular subtypes of breast cancer. Each dot represents one patient. Gene expression levels are presented as FPKM in log2 expression. Red dots are basal-like triple-negative (TN) breast cancer patients. Blue dotted line indicates KCNMA1 gene expression in normal breast cells. FPKM: Fragments Per Kilobase of transcript per Million

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  Fig. 2. Protein expression of BK channel α-subunit (forming the functional channel) in TNBC cells and patient tissues. a Western blots with antibody, M: marker, MDA231: MDA-MB-231 cells (cell positive control), Normal: normal primary breast tissue, TNBC: TNBC patient tissue, MB: mouse brain (tissue positive control). β-actin was used as a loading control. b Western blots with antibody and antigen, confirming signals detected by the antibody in (a). Figure 2a and b were prepared by cropping the full WB shown in Supplemental Fig. 15 by removing the white spaces above and below the signals of interest. c Quantitative expression levels of BK channel proteins normalized to β-actin (n = 4–6). Unpaired t test was performed. Two-tailed p = 0.0002, t = 0.8172. d Western blots using a BK channel protein antibody in MDA231, SUM159, MCF10A, and HCC1143, β-actin as loading controls. Figure 2d was prepared by cropping the full WB in Supplemental Fig. 16 by removing the white spaces above and below the signals of interest

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  Fig. 3. Biophysical properties of BK channels in MDA-MB-231 cells. a BK channel currents elicited by depolarizing pulses ranging from -40 mV to + 50 mV in 10 mV increment, pulse protocol is shown below. b BK channel conductance, G, − voltage relationship in the absence (black) and presence (red) of 0.1 μM IbTX (n = 8). c Effects of BMS-191011 and IbTX on membrane potential (Em) in MDA-MB-231

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  Fig. 4. BK channel openers induced cell death in TNBC cell lines. a BMS-191011 (20 μM) induced cell death on normal breast cells (MCF10A, dark), ER+ breast cancer cells (MCF7, grey), and TNBC cells (MDA-MB-231, red) (n = 5). One-way ANOVA was performed, p < 0.0001, F = 65.38. b Time- and concentration-dependent effects of NS11021 on growth of MDA-MB-231 cells (n = 6). Two-way ANOVA was performed, p < 0.0001, F = 66.67, DF = 9. c Time-dependent effect of BMS-191011 (20 μM) on growth of SUM159 cells (n = 4). Two-way ANOVA was performed, p < 0.0001, F = 618.9, DF = 6. d Time-dependent effect of BMS-191011 (20 μM) on HCC1143 cells (n = 4). Two-way ANOVA was performed, p < 0.0001, F = 27.6, DF = 4

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  Fig. 5. BMS-191011 induced activation of caspase-3 in MDA-MB-231. Using caspase-3/7 green dye, fluorescence was detected after incubation of 10 μM BMS-191011 for three days (b). Light image is shown in (a). Scale bar: 20 μm. Control cells are shown in the Supplemental Fig. 14. Using immunoblots, caspase-3 was detected in three TNBC cell lines (MDA-MB-231, HCC1143, SUM159) (c). Cleaved caspase-3 was detected after BMS-191011 treatment (d, M-marker, red arrow indicates pro-caspase 3, black arrow indicates cleaved active caspase-3). Figure 5c and d were prepared by cropping the full WB in Supplemental Figs. 17 and 18, respectively, by removing the white spaces above and below the signals of interest

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  Fig. 6. BMS-191011 induced arrest in S/G2 phases of MDA-MB-231. a: No treatment (control), b: Drug treatment for 24 h, c: Drug treatment for 48 h. Blue: G1 phase; Green: S phase; Red: G2 phase. Count: number of cells, PI-A: fluorescence intensity. Sub-G1 peak was gated out

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  Fig. 7. Slowing of MDA-MB-231 xenograft tumor by BK channel opener. a: Three paired-tumors of similar sizes (C1/T1, C2/T2, C3/T3) are shown to illustrate the inhibitory effects of BMS-191011 in tumor growth. Ultrasound imaging was performed once a week to calculate the tumor volume (mm3) for four weeks. A reconstruction of the tumor volume from ultrasound imaging is shown in the inset. b: Final average tumor volume in control (n = 8) and treated groups at week 8 (n = 8). *: p < 0.05 (t-Test was performed, two-tailed p = 0.0356, t = 2.32592)