Characterization of BKCa channels in murine and human BCCs.

(A; B) I-V curves (left) and corresponding maximal currents (right) of MMTV-PyMT WT (A) and MMTV-PyMT BK-KO cells (B), either under control conditions, or in the presence of paxilline or iberiotoxin. Data represents average ± SEM. N (cells) = 15 WT ctrl, 17 WT + PAX, 17 WT + IBTX, 16 BK-KO ctrl, 17 BK-KO + PAX, 19 BK-KO + IBTX. ***p≤0.001, Brown-Forsythe and Welch ANOVA test followed by Games-Howell’s multiple comparison test. ‡p≤0.001 compared to respective WT condition, Welch’s t-test. (C) Representative fluorescence images (left) and statistics (right) of MMTV-PyMT WT and BK-KO cells loaded with the ΔΨPM sensitive dye Dibac4(3). N = 6 independent experiments, **p≤0.01, Unpaired t-test. (D) I-V curves (left) and maximal currents (right) of MDA-MB-453 cells, either under control conditions, or in the presence of paxilline or iberiotoxin. Data represents average ± SEM. N (cells) = 30 ctrl, 22 + PAX, 24 + IBTX. ***p≤0.001, Kruskal-Wallis test followed by Dunn’s multiple comparison test. (E) I-V curves (left) and maximal currents (right) of MCF-7 cells, either under control conditions, or in the presence of paxilline or iberiotoxin. Data shows average ± SEM. N (cells) = 16 ctrl, 20 + PAX, 15 + IBTX. (F) Schematic representation of constructs used for over-expression in MCF-7 cells. The DEC exon is indicated in green. (G) Representative images (left) of MCF-7 cells either expressing BK RFP (upper) or BKCa-DECRFP (lower), additionally stained with MitoGREEN. Average Pearson correlation ± SEM of MitoGREEN and RFP of BKCa or BKCa-DEC are shown. N (cells) = 18 BKCa – RFP, 23 BKCa-DECRFP. **p≤0.01, Unpaired t-test. (H) I-V curves (left and middle) and corresponding maximal currents (right) of MCF-7 cells expressing BK RFP (left) or BK Ca-DECRFP (middle), respectively, either under control conditions, or in the presence of paxilline or iberiotoxin. Data represents average ± SEM. N (cells) = 18BK RFP ctrl, 14 BK RFP + PAX, 19 BK RFP + IBTX, 18 BK -DECRFP ctrl, 21 BK -DECRFP + PAX, 18 BKCa-DECRFP + IBTX. **p≤0.01, ***p≤0.001, Brown-Forsythe and Welch ANOVA test followed by Games-Howell’s multiple comparison test. †p≤0.01 between ctrl conditions, Welch’s t-test.

BKCa modulates the subcellular Ca2+ homeostasis in BCCs.

Cytosolic (A – C), endoplasmic reticulum (ER) (D – F), and mitochondrial Ca2+ dynamics (G – I) over-time of MMTV-PyMT WT and MMTV-PyMT BK-KO cells (A, D, G), MDA-MB-453 cells (B, E, H) or MCF-7 cells (C, F, I). All data represent average ± SEM. At time points indicated in the panels, cytosolic and mitochondrial Ca2+ alterations were evoked by extracellular stimulation with ATP (A – C, G – I), or by Ca2+ depletion of the ER using Ca2+-free buffer containing the Ca2+ chelator EGTA, followed by administration of the SERCA inhibitor BHQ prior to ATP administration (D – F). MMTV-PyMT (A, D, G) and MDA-MB-453 (B, E, H) cells were either measured under control conditions, or in the presence of paxilline (+ PAX) or iberiotoxin (+ IBTX). MCF-7 cells (C, F, I) either expressed an RFP (ctrl), BK RFP, or BK Ca-DECRFP. N (independent experiments) = A: 17 WT ctrl, 18 BK-KO ctrl, 6 WT + PAX and BK-KO + PAX, 5 WT + IBTX and BK-KO + IBTX, B: 4 for all, C: 5 for all, D: 14 WT ctrl, 13 BK-KO ctrl, 8 WT + PAX and BK-KO + PAX, 6 WT + IBTX and BK-KO + IBTX, E: 7 ctrl, 9 + PAX, 5 + IBTX, F: 4 for all, G: 11 WT ctrl, 12 BK-KO ctrl, 6 WT + PAX and BK-KO + PAX, 5 WT + IBTX, 4 BK-KO + IBTX, H: 8 ctrl and + PAX, 5 + IBTX, i: 5 ctrl, 4 + BKCaRFP and BKCa-DECRFP. **p≤0.01, ***p≤0.001. Kruskal-Wallis test followed by Dunn’s MC test (A, B, C, D, I), One-Way ANOVA test followed by Tukey’s MC test (E, F, G) or Brown-Forsythe ANOVA test followed by Games-Howell’s MC test (H). #p≤0.05, ‡p≤0.001 compared to respective WT condition, Mann-Whitney test (A, D, ctrl in G), or Welch’s t test (+ IBTX in G).

BKCa channels promote the metabolic activity of BCCs.

(A, D) Average ECAR over-time ± SEM of MMTV-PyMT WT (A, red) and BK-KO cells (A, black), or MDA-MB-453 cells (D) in response to administration of Oligomycin-A (+O), FCCP (+F) and Antimycin-A (+A) at time points indicated. (B, E) Average basal and (C, F) maximal ECAR ± SEM of MMTV-PyMT WT (B, C, left) and BK-KO cells (B, C, right), or MDA-MB-453 cells (E, F) under control conditions, or in the presence of paxilline or iberiotoxin. (G, J) Average OCR over-time ± SEM of MMTV-PyMT WT (G, red) and BK-KO cells (G, black), or MDA-MB-453 cells (J) in response to administration of Oligomycin-A (+O), FCCP (+F) and Antimycin-A (+A) at time points indicated. (H, K) Average basal and (I, L) maximal OCR ± SEM of MMTV-PyMT WT (H, I, left) and BK-KO cells (H, I, right), or MDA-MB-453 cells (K, L) under control conditions, or in the presence of paxilline or iberiotoxin. (M – P) LC-MS-based determination of major glycolytic and mitochondrial metabolites in particular Lactate (M, [Lactate]), Pyruvate (N, [Pyruvate]), Asparagine (O, [Asparagine]) and Glutamine (P, [Glutamine]) of MMTV-PyMT WT (left panels), BK- KO (middle panels) and MDA-MB-453 cells (right panels), either under control conditions or after cell cultivation with paxilline. N (independent experiments) = A, B, C, G, H, I: 7 WT ctrl and BK-KO, 3 for all others, D, E, F, J, K, L: 3 for all, M – P: 7 for BK-KO ctrl, 6 for all others. *p≤0.05, **p≤0.01, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s MC test (B, E, F, I), Brown-Forsythe and Welch ANOVA test followed by Games-Howell’s MC test (C, H), One-Way ANOVA test followed by Tukey’s MC test (K, L) or Mann-Whitney test (M – P). #p≤0.05, †p≤0.01, ‡p≤0.001, to respective WT condition, Mann-Whitney test (B, C, + IBTX in I, M – P), Welch’s t-test (ctrl in H and I) or Unpaired t-test (+ IBTX in H).

Expression of BKCa modulates mitochondrial function and glucose uptake of BCCs.

(A – D) Representative fluorescence images and -ratios (Fmito/Fnuc) over-time (A, C), and corresponding statistics ± SEM (B, D) representing ΔΨmito of TMRM-loaded MMTV-PyMT WT and BK-KO (A, B) and MDA-MB-453 cells (C, D) under basal conditions (A, C, upper images) and upon administration of FCCP for mitochondrial depolarization (A, C lower images). (E – J) [ATP]mito dynamics ± SEM over-time of MMTV-PyMT WT and BK-KO cells (E), MDA-MB-453 cells (G) and MCF-7 cells (I) in response to extracellular glucose removal (left panels) or upon administration of Oligomycin-A (right panels). F, H and J show changes of [ATP]mito induced by glucose removal to Oligomycin-A administration ± SEM, under control conditions, or in the presence of paxilline or iberiotoxin (F, H), or upon expression of BK RFP or BK -DECRFP (J). (K – M) Basal mitochondrial H O concentrations ± SEM of MMTV-PyMT WT (K, left), BK-KO (K, right), MDA-MB-453 (L) and MCF-7 cells (M), either under control conditions, in the presence of paxilline or iberiotoxin (K, L), or upon expression of BK RFP or BK -DECRFP (M). (N, P) Representative fluorescence wide-field images (left) and corresponding statistics ± SEM (right) of MMTV-PyMT WT (N, left images and red bars) and BK-KO cells (N, right images and black bars) or MDA-MB-453 cells (P) incubated with 2-NBDG, either in the absence (upper images) or presence of FCCP (lower images). (O, Q) Average ± SEM of FCCP induced change in 2-NBDG uptake of MMTV- PyMT WT (O, left) and BK-KO cells (O, right), or MDA-MB-453 cells (Q) either under control conditions, or in the presence of paxilline or iberiotoxin. Values above 1 indicate that mitochondria prevent, values below 1 that mitochondria support glucose uptake. N (independent experiments) = A – D, N – Q: 4 for all, E – J [- Glucose]: 8 WT ctrl, BK-KO ctrl and BK-KO + IBTX, 7 WT + IBTX, 6 WT and BK-KO + PAX and MCF-7 ctrl, 3 MDA-MB-453 + PAX, 5 for all others. E – J [+ Oligomycin-A]: 11 WT ctrl, 8 BK-KO ctrl and MDA-MB-453 + IBTX, 7 WT + PAX and WT + IBTX, 6 BK-KO + PAX, 3 MDA-MB-453 + PAX and 5 for all others. *p≤0.05, **p≤0.01, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s MC test (B, D, F, H, J, K, O), Brown-Forsythe and Welch ANOVA test followed by Games- Howell’s MC test (L, M), Mann-Whitney test (N), Unpaired t-test (P) or Welch’s t-test (Q). #p≤0.05, †p≤0.01, ‡p≤0.001, to respective WT condition, Mann-Whitney test (B, F, + PAX and + IBTX in K, ctrl in O), Unpaired t-test (ctrl in K, + PAX and + IBTX in O).

BKCa activity is present in the inner mitochondrial membrane (IMM) of BCCs.

(A) Representative BKCa single-channel recordings of the IMM of mitoplasts isolated from MDA-MB-453 cells using a symmetric 150/150 mM isotonic KCl solution containing 100 µM Ca2+ at voltages ranging from -80 to +80 mV as indicated in the panel. (B) Open probability analysis of mitoBKCa at different voltages received from experiments as performed in (A). N = 8. (C) Single-channel currents of the IMM of mitoplasts isolated from MDA-MB-453 cells recorded using a voltage ramp protocol ranging from −150 to +150 mV. Above and below the ramp are enlarged excerpts of the records shown in rectangles. (D) Current-voltage (I-V) plot based on single-channel recordings of MDA-MB-453 cells as performed in a, using a symmetric 150/150 mM KCl isotonic solution containing 100 µM Ca2+. N = 11. (E, F) Representative single-channel recordings of the IMM of mitoplasts isolated from MDA-MB-453 cells (E) and corresponding open probabilities at +40 mV in a symmetric 150/150 mM KCl isotonic solution under control conditions (100 μM Ca2+), after reducing Ca2+ to 1 μM, re-addition of 100 μM Ca2+ and finally after application of 5 μM paxilline in the presence of 100 μM Ca2. Data in (F) show average ± SEM. *p≤0.05, **p≤0.01 using Friedmann test followed by Dunn’s multiple comparison test, n =7. (G) Pie chart displaying the percentage of mitoBKCa channel currents (green) possessing a conductance of ∼210 pS, versus the total number of patch-clamp experiments performed using mitoplasts isolated from MDA-MB- 453 cells (upper left), MMTV-PyMT WT cells (upper right), MCF-7 cells (lower left) and MMTV-PyMT BK-KO cells (lower right). Black segments represent empty patches, bright- and dark grey fraction demonstrate percentage of channels possessing smaller conductances of ≤100 pS and ≤150 pS, respectively. All recordings were low-pass filtered at 1 kHz. “c“ and “o” indicate the closed- and open state of the channel, respectively.

BKCa-DEC expression contributes to the metabolic remodeling and growth of murine and human BCCs and is present in primary tumor samples.

(A) Schematic representation of the fate of glucose in glycolysis. The tricarboxylic acid (TCA) cycle or lactate secretion via monocarboxylate transporters (MCT) can be inhibited, either using NaN3 or BAY-8002. GLUT: Glucose transporter, HKs: Hexokinases. (B) Representative cytosolic lactate concentration ([LAC]cyto) of a MMTV-PyMT WT cell over-time in response to administration or removal of NaN3 and BAY-8002 at time points indicated. Dashed lines indicate slopes taken for assessment of the “Warburg index. (C – G) Average Warburg indices ± SEM of MMTV-PyMT WT (C, F, left), MMTV-PyMT BK-KO (C, F, right), MDA-MB-453 cells (D, G) and MCF-7 cells (E) calculated from the experiments as shown in (B), either under control conditions, in the presence of paxilline or iberiotoxin (C, D), upon expression of BK RFP or BK Ca-DECRFP (E), or upon cell treatment with a scrambled siRNA (siScrbl), or siRNA against a common BKCa sequence targeting all known splice variants (siBK), or a siRNA specifically designed to knockdown BKCa-DEC (siDEC) (F, G). (H, I) Normalized MTT absorbance over-time of MMTV-PyMT WT (H, left) and BK- KO cells (H, right), and MDA-MB-453 cells (I), either under control conditions, or in the presence of paxilline or iberiotoxin. j, Representative images and corresponding statistics of colony formation assays using MMTV-PyMT WT or BK-KO cells in the presence or absence of O2. (K – N) mRNA expression of BKCa and BKCa-DEC as performed by Nanostring analysis of 551 BC patient samples. (K) Log2 expression counts of BKCa and BKCa-DEC. The threshold for positive expression level was set to log2 = 5.5 (dashed line). (L) Log2 expression counts of BKCa-DEC blotted over the log2 expression counts of BKCa. 10 of the 551 patient samples showed expression of BKCa-DEC above the threshold of log2 = 5.5 (dashed line), whereas 541 patient samples were BKCa-DEC negative. (M) Correlation of the log2 expression counts of BKCa-DEC positive samples with the log2 expression counts of BKCa in the primary human BC material. (N) Summarizing scheme of BKCa in cancer cell homeostasis. N (independent experiments) = A – E: 4 WT and BK-KO ctrl (A), 6 WT + PAX (C), 5 BK-KO + PAX (C), WT siScrbl and WT siBK (F) and MDA-MB-453 + PAX (D), MCF-7 + BKCa – RFP (E), 4 WT and BK-KO + IBTX (C), 7 for all others. F, G: 4 WT siDEC, 5 for all others. H – J: 4 for all. *p≤0.05, **p≤0.01, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s MC test (C, E, F, G, I), One-Way ANOVA test followed by Tukey’s MC test (H) or Mann-Whitney test (D). †p≤0.01, ‡p≤0.001 compared to respective WT condition, Unpaired t-test (ctrl, C, J) or Mann-Whitney test (+ IBTX, C, F).

Representative whole-cell patch-clamp traces and colocalization analysis in breast cancer cells.

(A – D) Representative whole-cell patch-clamp traces of MMTV- PyMT WT (A), MMTV-PyMT BK-KO (B), MDA-MB-453 (C) and MCF-7 wild-type cells (D), either under control conditions (ctrl), or in the presence of 5 µM paxilline (+ PAX) or 30 nM iberiotoxin (+ IBTX), respectively, as indicated in the panels. (E) Representative images (left) of MCF-7 cells either expressing a mitochondrial targeted red fluorescent protein (mtRFP, upper images, second column) or a red fluorescent protein fused to a glycosylphosphatidylinositol (GPI)- anchor (RFP-GPI, lower images, second column). Cells were additionally stained with MitoGREEN for visualization of mitochondria (first column). Merge of the channels and a zoom are demonstrated. Right panel shows average Pearson correlation ± SEM of MitoGREEN and RFP of mtRFP (left bar) or RFP-GPI (right bar). Grey dashed lines indicate average colocalization scores of MitoGREEN and RFP of mtRFP and RFP-GPI, which is also indicated in Figure 1g. N (cells) = 24 for mtRFP and 12 for RFP-GPI. ***p≤0.001 Unpaired t-test. (F, G) Representative whole-cell patch-clamp traces of MCF-7 cells either expressing BKRFP (F) or BK Ca-DECRFP (G) under control conditions (ctrl, left panels), or in the presence of 5 µM paxilline (+ PAX, middle panels) or 30 nM iberiotoxin (+ IBTX, right panels). (H) Global cellular RFP-fluorescence intensities of MCF-7 cells used for patch-clamp experiments. Cells either expressed BKRFP (red bar) or BK Ca-DECRFP (green bar). Data represents average ± SEM of 18 cells for both conditions.

Cytosolic-, ER-, and mitochondrial Ca2+ homeostasis is altered by functional BKCa expression in breast cancer cells.

(A, B) Fura-2 ratio signals (F340/F380) of MMTV- PyMT WT or BK-KO cells over-time in response to cell stimulation with 100 µM ATP. Experiments were either performed in the presence of 5 µM paxilline (A) or 30 nM iberiotoxin (B). (C – E) Maximal fura-2 ratio signals of MMTV-PyMT WT and BK-KO (C), MDA-MB-453 (D) or MCF-7 cells (E) upon stimulation with 100 µM ATP, under control conditions, in the presence of 5 µM paxilline or 30 nM iberiotoxin (C, D), or upon expression of BK RFP or BK -DECRFP (E). (F, G) FRET-ratio signals over-time of MMTV-PyMT WT and BK-KO cells expressing D1ER, a FRET- based ER Ca2+ sensor. Throughout the experiments, either 5 µM paxilline (F) or 30 nM iberiotoxin (G) were present. (H, I) [Ca2+]mito over-time of MMTV-PyMT WT or BK-KO cells in response to cell stimulation with 100 µM ATP. [Ca2+]mito was assessed using 4mtD3cpV, a FRET-based Ca2+ indicator targeted to the mitochondrial matrix. Experiments were either performed in the presence of 5 µM paxilline (H) or 30 nM iberiotoxin (I). (J – L) Maximal FRET-ratio (FRET/CFP) signals received upon stimulation of MMTV-PyMT WT or BK-KO (J), MDA-MB-453 (K) or MCF-7 cells (L) with 100 µM ATP. Experiments were either under control conditions, in the presence of 5 µM paxilline or 30 nM iberiotoxin (J, K), or upon expression of BKRFP or BK -DECRFP (L). All data represent average ± SEM. N (independent experiments) = A, B: 6 WT + PAX and BK-KO + PAX, 5 WT + IBTX and BK-KO + IBTX. C – E: 17 WT ctrl, 18 BK-KO ctrl, 6 WT + PAX and BK-KO + PAX, 5 WT + IBTX, BK-KO + IBTX and all MCF-7, 4 for all MDA-MB-453. F, G: 8 WT + PAX and BK-KO + PAX, 6 WT + IBTX and BK-KO + IBTX. H, I: 6 WT + PAX and BK-KO + PAX, 5 WT + IBTX, 4 BK-KO + IBTX. J – L: 11 WT ctrl, 12 BK-KO ctrl, 6 WT + PAX and BK-KO + PAX, 5 WT + IBTX, MDA-MB-453 + IBTX and all MCF-7, 4 BK-KO + IBTX, 8 MDA-MB-453 ctrl and + PAX. *p≤0.05, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s MC test (C), One- Way ANOVA test followed by Tukey’s MC test (E, J) or Brown-Forsythe and Welch ANOVA test followed by Games-Howell’s MC test (K). ‡p≤0.001 compared to respective WT condition, Mann- Whitney test (ctrl in C, J), Welch’s t-test (+ PAX in C). Unpaired t-test (+ IBTX in J).

Paxilline and iberiotoxin differentially modulate ECAR and OCR in MMTV-PyMT WT and BK-KO cells.

(A, B) Average ECAR over-time ± SEM of MMTV-PyMT WT (bright red and salmon) and BK-KO cells (dark and bright grey) in response to administration of Oligomycin-A (+O), FCCP (+F) and Antimycin-A (+A) at time points indicated in the panel. Experiments were either performed in the presence of 5 µM paxilline (A) or 30 nM iberiotoxin (B). N = 3 for all. (C, D) Average OCR over-time ± SEM of MMTV-PyMT WT (bright red and salmon) and BK-KO cells (dark and bright grey) in response to administration of Oligomycin-A (+O), FCCP (+F) and Antimycin-A (+A) at time points indicated in the panel. Experiments were either performed in the presence of paxilline (C) or iberiotoxin (D). N = 3 for all. (E) Schematic representation of the workflow for LC-MS-based metabolomics. HILIC: Hydrophilic interaction liquid chromatography

BKCa modulates cellular substrate dependency for maintaining [ATP]mito and reverses FOF1 ATP-synthase.

(A) Average fluorescence ratios (Fmito/Fnuc) of MMTV- PyMT WT and BK-KO cells, either in the presence of 2.0 mM or 25.0 mM extracellular glucose. N = 5 BK-KO 2.0 mM GLU, 6 for all others. ***p≤0.001, Mann-Whitney test. †p≤0.01, ‡p≤0.001 compared to 2.0 mM glucose condition of the respective cell type, Mann-Whitney test. (B – E) Average changes in FRET-ratio signals ± SEM induced either upon extracellular glucose removal (B, D) or upon administration of Oligomycin-A (C, E) to MMTV-PyMT WT, BK-KO (B, C) or MDA- MB-453 cells (D, E) expressing mtAT1.03, a FRET-based ATP sensor targeted to the mitochondrial matrix. Experiments were either performed under control conditions or in the presence of 5 µM paxilline or 30 nM iberiotoxin. N’s for glucose removal (-Glucose) experiments = 8 WT and BK-KO ctrl, 6 WT and BK-KO + PAX, 7 WT + IBTX, 5 BK-KO + IBTX, 5 MDA-MB-453 ctrl, 3 MDA-MB-453 + PAX, 5 MDA-MB-453 + IBTX. N’s for Oligomycin-A administration (+Oligomycin-A) experiments = 11 WT ctrl, 8 BK-KO ctrl, 7 WT + PAX and WT + IBTX, 6 BK-KO + PAX, 8 BK- KO + IBTX, 5 MDA-MB-453 ctrl, 3 MDA-MB-453 + PAX and 8 MDA-MB-453 + IBTX. *p≤0.05, **p≤0.01, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s MC test (B, C, D). #p≤0.05, ‡p≤0.001 compared to respective WT condition, Mann-Whitney test (ctrl in B, all in C), or Welchs t-test (+ IBTX in B). (F, G) Average changes in FRET-ratio signals ± SEM induced either upon extracellular glucose removal (F) or upon administration of Oligomycin-A (G) to MCF-7 cells expressing mtAT1.03, either in combination with a red fluorescent protein as control, BKCa BKCa-DECRFP. *p≤0.05, **p≤0.01, Kruskal-Wallis test followed by Dunn’s MC test. N = 6 MCF-7 ctrl – glucose, 5 for all others. (H) Schematic representation of processes involved in 2-NBDG uptake. 2-NBDG is taken up via glucose transporters (GLUTs, green), and ATP-dependently phosphorylated by hexokinase isoforms (HKs, red) to 2-NBDG-6-phosphate (2-NBDG-6P). Under basal conditions, if FOF1 ATP-synthase is running in forward mode (left panel), mitochondria contribute to 2-NBDG uptake by ATP generation and delivery to HKs via the adenine nucleotide transporter (ANT, yellow), and the voltage-dependent anion channel (VDAC, violet). Contrary, under basal conditions, if FOF1 ATP-synthase activity is reversed (right panel) it competes with HKs for ATP. Subsequent inhibition of mitochondria due to their depolarization with FCCP (red) stops ATP-synthase activity. Under these conditions, 2-NBDG uptake will decrease if FOF1 ATP-synthase operates in forward mode (left panel), but will increase if FOF1 ATP-synthase shows reversed activity (right panel). (J – L) Average fluorescence signal (a.u.) ± SEM of MMTV-PyMT WT and BK-KO cells (J, K), or MDA-MB-453 cells (L) incubated with 100 µM 2-NBDG at 37°C for 30 minutes either under control conditions (J) or in the presence of 200 nM FCCP for mitochondrial depolarization (K, L), with or without 5 µM paxilline or 30 nM iberiotoxin. N = 4 for all. *p≤0.05, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s MC test (J, and WT in K), One-Way ANOVA test (BK-KO in K) or Welch’s t-test (L). #p≤0.05, ‡p≤0.001 compared to respective WT condition, Mann-Whitney test (ctrl and + IBTX in J, ctrl in K), or Unpaired t-test (+ PAX in J and + PAX in K).

BKCa is present in the inner mitochondrial membrane of MMTV- PyMT WT and MDA-MB-453 cells.

(A – C) Graphs show representative BKCa single-channel recordings of the inner mitochondrial membrane of mitoplasts isolated from MMTV-PyMT WT cells using a symmetric 150/150 mM isotonic KCl solution, either containing 100 µM Ca2+ (A), 1 µM Ca2+ (B), or 5 µM Paxilline in the presence of 100 µM Ca2+ (C), at voltages ranging from -80 to +80 mV as indicated in the panels. The patch in a contained two BKCa channels. “c“ indicates the closed-, “o” the open state of the channel. (D) Representative western blot of BKCa (upper), Na+/K+ ATPase as a plasma membrane marker (middle), and cytochrome c oxidase subunit IV (COXIV) as a mitochondrial marker (lower). Western blot was performed using whole-cell lysates (L), the homogenate (H), crude isolated mitochondria (C) and mitochondria after percoll gradient purification (P) of MDA-MB-453 cells. N = 3.

siRNA treatment reduces expression of BKCa and BKCa-DEC, and BKCa-DEC decreases dependency on oxidative metabolism in breast cancer cells.

(A) Schematic representation of siRNAs used for subsequent silencing experiments. Either an siRNA targeting all known BKCa isoforms, referred to as siBK (orange), or an siRNA specifically designed against the DEC exon (green), referred to as siDEC (violet), were used. (B, C) Relative mRNA expression levels ± SEM of BKCa and BKCa-DEC in MMTV-PyMT WT (B) and MDA-MB-453 cells (C), as analysed by qPCR. Cells were either treated with a scrambled siRNA as a control (siScrbl), siRNA against all known BKCa isoforms (siBK) or siRNA specifically targeting BKCa-DEC (siDEC), respectively. N = 4 siBK MMTV-PyMT WT, 3 for all others. (D) Normalized colony sizes of colony formation assays performed using MMTV-PyMT WT and BK-KO cells. Cells were cultivated for 7 days either in the presence or absence of O2. *p≤0.05, Mann-Whitney test. N = 4 independent experiments for all conditions.

Probes used for Nanostring nCounter gene expression analysis.

Primers used for qPCR analysis.

siRNAs used for silencing based experiments.