KCNMA1 gene expression in The Cancer Genomic Atlas (TCGA) database
KCNMA1 gene expression patterns in primary breast cancer database were analyzed from The Cancer Genome Atlas (TCGA) [18] via cBioPortal (http://cbioportal.org ) [19]. Gene expression levels from RNA-sequencing data was illustrated in log2-fold of fragments per kilobase of transcript per million (FPKM) [20].
Cell culture and plasmid transfection
Human breast adenocarcinoma cells (ER+ MCF7, triple-negative MDA-MB-231/Luc and SUM159) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), whereas triple-negative HCC1143 cells were grown in RPMI (Invitrogen), supplemented with 10% fetal bovine serum, 1X Pen/Strep (Gbico 15140). Normal human mammary epithelial cells (MCF10A) were grown in mammary epithelial cell basal medium (MEC), supplemented with MEC growth kit (ATCC). Rat h9c2 cardiac myocytes were purchased from ATCC (CRL-1446), cultured in DMEM. All human cell lines were obtained five years ago from American Type Cell Collection (ATCC) in which authentication was performed using immunoblots. ATCC catalog numbers are CRL-10317 for MCF10A, HTB-22 for MCF7, HTB-26 for MDA-MB-231, and CRL-2321 for HCC1143. SUM159 was a gift from Dr. Elena Pugacheva (co-author) that was authenticated using immunoblots. We did not reauthenticate MCF10A, MCF7, MDA-MB-231, and HCC1143. All cell lines used in this study were tested negative for mycoplasma contamination using Mycoplama Detection Kit (InvivoGen, PlasmoTest, Cat#: rep-pt1).
Cells with 50-70% confluence in 6-well plates were used for transient plasmid (1-2mg) transfection using Lipofectamine3000 transfection reagent (Invitrogen). Human wild type (hSlo1 WT) and A313D mutant (hSlo1-A313D) channel cDNA in pcDNA3 mammalian expression vector were co-transfected with GFP for verification of expression.
Patch clamp studies in MDA-MB-231
Details in whole-cell or perforated (or permeabilized) patch clamp studies in isolated cells have been previously reported [21, 22]. Briefly, the cells grown on coverslips were placed in a lucite bath with the temperature maintained at 35oC - 37oC. Em and voltage-gated potassium currents were recorded using the whole cell patch clamp technique with an Axopatch‑700B amplifier. Em was measured with DMEM or Tyrode solution and pipette solution. DMEM contained physiological ion concentration (in mM): 150Na+, 5K+, 2.0Ca2+ (pH = 7.4). Tyrode solution contains (mM): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1, Glucose 5.5, Hepes 5, pH 7.4 adjusted by NaOH. The pipette solution contained (in mM): 85 KCl, 40 K-Aspartate, 0.1CaCl, 10 HEPES, pH was adjusted to 7.2 by KOH. The pipettes had a resistance of 2–5 MW when filled with pipette solution. For perforated patch, amphotericin - B was added to the pipette solution to a final concentration of 240mg/ml. The whole-cell/perforated patch clamp data were acquired by CLAMPEX and analyzed by CLAMPFIT (pClamp 9, Axon/Molecular Device).
Live cell imaging
Live cell imaging experiments were performed using a Zeiss Axio Observer A1 inverted microscope with fluorescence. Images were acquired and analyzed using AxioVision (version 4.6). We used ethidium homodimer-1 (EthD-1, Invitrogen, 0.2-0.5ml of 2mM stock to 1ml culture of cells in 6-well plates) to label dead cells (Fig.4; Supplemental Figures 3-7). Ethidium homodimer assay was utilized to detect dead cells. Ethidium homodimer is impermeable to the membrane of a living cell. However, when the cell dies the ethidium homodimer fluorescent dye is able to bind to the DNA, emitting bright red fluorescent signals. Cell count was performed using ImageJ (NIH).
Western blotting
Cells were harvested using Radioimmunoprecipitation Assay (RIPA) buffer with 1% protease inhibitor cocktail (Sigma). We then sonicated lysates on ice and centrifuged at 12,000xg for 10 minutes at 4oC. Tumor tissue was homogenized in RIPA buffer with 1% protease inhibitor cocktail (Sigma), then centrifuged at 12,000xg for 10 minutes at 4oC. Supernatant was isolated from debris pellet.
Protein concentration was measured using Bicinchoninic acid assay (BCA) (Thermo Fisher). Once protein concentrations were normalized across samples, they were then heated for 12 minutes at 90oC. Samples were loaded into NuPage 4–12% bis-tris gels (Invitrogen) with MOPS running buffer at 70 volts for 100 minutes, then transferred to 0.2 mm pore PVDF membrane (Thermo Scientific) at 30 volts for one hour in cold room. Next, blots were blocked in Licor blocking buffer for one hour, and incubated for 12 hours at 4 degrees with primary antibody for either anti-KCNMA1 for epitope 199-213 (Alomone cat# APC-151), anti-caspase-3 (Cell Signaling Technology), or anti-Slo1 for c-terminus segment 9-10 (Millipore) at 1:500 dilution. The membrane was then washed 3 times for 15 minutes with tris-buffered saline containing 0.1% tween-20 (TBS-T). A secondary antibody, IR 800 CW from Licor (1:20,000 dilution) was incubated with the membrane at room temperature for one hour. After three 10-minute washes with TBS-T, blots were imaged using a Licor Odyssey CLx and image studio software. If residual background signal was observed, additional washes of 5 to 10 minutes with TBS-T were completed and the membrane was re-imaged. Beta-actin primary antibody (Proteintech) and IRDye 680RD secondary antibody (Licor) were used as a loading control.
Immunohistochemistry of patient breast samples
Experiments involving patient breast samples were approved by West Virginia University Institutional Review Board. Formalin-fixed paraffin-embedded (FFPE) breast tumor tissue from patients was processed according to vendor’s manual instruction (Biocare) and following a verified protocol in the Pathology Laboratory of Translational Medicine at WVU. Briefly, 3mm sections were deparaffinized on slides, quenched with hydrogen peroxide, and incubated in BK channel antibody (Sigma-Aldrich, HPA054648) Sigma at 4oC for 4 minutes. Horseradish peroxidase-containing secondary antibody (UMap anti-RB, Roche, Diagnostic, Cupertino, CA) was then added for 8 minutes and developed using Biocare DAB (brown color). Hematoxylin was used as a counterstain (blue color).
Automated formalin-fixed, paraffin embedded immunohistochemical staining, to evaluate tumor antigen expression profiles, is available via the Ventana DISCOVERY automated IHC Platform. Breast tumor IHC slides stained with BK channel antibody were examined under an Olympus VS-120 slide scanner with a 10X Plan S Apo/0.40 NA objective, equipped with a color camera (Pike 505C VS50). The images were analyzed using OlyVIA (Olympus) and ImageJ (NIH). Percentage of area staining was used to quantify the protein expression levels in IHC slides.
Breast tumor induction in NOD scid gamma (NSG) mice
Female NSG-immunodeficient mice of 4-6 weeks old were purchased from the Jackson Laboratory. Experimental procedures and housing of the animals were approved by the Institutional Animal Care and Use Committee. Animal were housed in a fully state-of-the-art facility that includes large specific pathogen free rooms, husbandry conditions (breeding program, light/dark cycle, temperature control, quality water, clean cages access to food and water), and welfare-related policies related to tumor studies (e.g., tumor burden policy).
Following power analysis, a total of 16 female mice was used, 8 for treatment, 8 for controls. For mammary pad injections, pathogen-free luciferase-expressing human breast adenocarcinoma cells (MDA-MB-231/Luc, 1-2x106 cells/animal) were injected into the fourth inguinal mammary gland of 6- to 8-week-old mice. Primary tumors had formed typically two weeks following cell injection. Tumor size was monitor by imaging twice a week. After experiment, mice were euthanized by isoflurane overdose (5% to effect or an overdose 100mg/kg of sodium pentobarbital), a procedure approved by our IACUC.
Ultrasound imaging of xenograft tumor in NSG mice
Details for ultrasound imaging of xenograft tumor in NSG mice have been previously reported [21]. Briefly, animals were anesthetized by exposure to 1-3% isoflurane during imaging. Imaging was performed weekly over the course of each experiment, typically for 4-6 weeks. Tumor volume was imaged by ultrasound imaging (USI) with Vevo2100 Micro-Ultrasound System. A 40 or 50 mHz transducer was used, depending on the tumor volume. A 3-dimensional (3D) image was acquired with scanning distance of 0.071 mm between images. Vevo software then integrated the images into a reconstructed 3D tumor from which the tumor volume was obtained.
BK channel openers
BMS-191011 (Tocris) and NS11021 (Tocris) were prepared in 10mM DMSO stock. The working concentrations of 10-50mM contained 1-5ml of DMSO in 1 mL DMEM medium, resulting in 0.1-0.5% of DMSO, which did not affect TNBC cells (Figure S7). For testing in vivo effects of MDA-MD-231, 3ml of 10mM (equivalent to 186.82ng) stock was added to 1mL PBS, 50ml was administrated directly into the xenograft grown in mouse via during day time in the animal imaging facility. For testing adverse effects of the drug, tail-vein injection was used.
Scratch (or “wound healing”) assay
MDA-MB-231 cells were incubated in a 24-well plate. After reaching confluence, the scratch (or “wound”) was created using a sterile 200-ml pipette tip, defined by the space within two red lines (upper left), filled (or “healed”) by migration of cells (upper right). Curved red line indicates the marker (shadowed area) used to identify the location of the scratch. Each petri dish reached 70% confluence before performing the assay. The difference between the control (untreated) and treated cell growth was visually demonstrated by less than 10 live cells in the BMS-191011 treated “wound” region (Supplemental Figure 10D), and a complete repopulation in the control (Supplemental Figure 10B).
Caspase-3/7 Green Fluorescence Dye
CellEvent Caspase-3/7 Green Detection Reagent was obtained from Thermo Fisher (Cat#: C10723). Working solution of 1mM was used in cell culture. Fluorescence microscopy was used to acquire images. Green fluorescence was detected only apoptotic cells.
Cell cycle analysis using flow cytometry
Cells were grown to 60-80% confluency in DMEM before drug treatment. Cells were either treated with BMS-191011 in DMSO, DMSO alone, or no treatment. After 24-48 hours, cells were washed with PBS and incubated with 0.25% trypsin with EDTA (Invitrogen) for 5 minutes at 37oC. After combining the resultant solution with 10mL PBS in 15mL tubes, cells were pelleted at 1000rpm for 6 minutes at 4oC. Cell pellets were resuspended in 200mL PBS then added with swirling to 2mL ice cold 70% ethanol. These single-cell suspensions were kept at 4oC until further processing.
For flow cytometry, cells were re-pelleted at 1000rpm for 6 minutes before decanting ethanol. After resuspension in 2mL PBS and incubation for 1 minute at room temperature, cells were pelleted and resuspended in 100mL of 0.2% Tween-20 in PBS at room temperature then incubated at 37oC for 15 minutes. Next, 100mL of PBS containing 2% fetal bovine serum and 0.1% sodium azide was added and cells were pelleted. The solution was then decanted, 10mL of RNase A (DNase- and protease-free; Thermo Scientific EN0531) in PBS (180mg/mL) was added and allowed to sit for 15 minutes at room temperature, and 20mL of propidium iodide (PI) in PBS (50mg/mL) was added for 15 minutes. The resultant solution was brought to a volume of 300-500mL using PBS before data collection.
Samples were analyzed on BD LSRFortessa using BD FACS Diva version 8.0 software. A minimum of 20,000 cells were collected for each sample. Data analysis was done using FCS Express 6.0 flow cytometry software (De Novo Software, Los Angeles CA). Cell cycle fit algorithm was selected using the lowest relative chi square value and BAD value < 20%. The sub-G1 peak in DNA profile plots was gated out to focus on altered distribution of G1/S/G2.
Statistical analysis
Data were shown as mean ± standard deviation (SD) in the text. Bar figures were presented as mean ± SD using GraphPad (Prism). Student’s t-test and two-way ANOVA (for more than two groups) were used for statistical analysis. P<0.05 was considered as statistically significant. Details in statistical analysis, such as F values and degree of freedom (DF) when using ANOVA and t values when using t-test, are included in the figure legends.