Figure 1. Characterization of BK_Ca 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 (B) cells, 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 ΔΨ_PM 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_Ca^RFP (upper) or BK_Ca-DEC^RFP (lower), additionally stained with MitoGREEN. Average Pearson correlations ± SEM of MitoGREEN and RFP of BK_Ca or BK_Ca-DEC are shown. n (cells) = 17 BK_Ca-RFP, 22 BK_Ca-DEC^RFP. *p≤0.05, Unpaired t-test. (H) I-V curves (left and middle) and corresponding maximal currents (right) of MCF-7 cells expressing BK_Ca^RFP (left) or BK_Ca-DEC^RFP (middle), respectively, either under control conditions, or in the presence of paxilline or iberiotoxin. Data represents average ± SEM. n (cells) = 18 BK_Ca^RFP ctrl, 14 BK_Ca^RFP +PAX, 19 BK_Ca^RFP +IBTX, 18 BK_Ca-DEC^RFP ctrl, 21 BK_Ca-DEC^RFP +PAX, 18 BK_Ca-DEC^RFP +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.

Figure 1—figure supplement 1. Representative whole-cell patch-clamp traces and colocalization analysis in BCCs.

(A, B) Representative whole-cell patch-clamp traces of MMTV-PyMT WT (A) and MMTV-PyMT BK-KO cells (B), 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. (C) Resting membrane potential (RMP) ± SEM of MMTV-PyMT WT (red) and BK-KO cells (black) in mV as measured using the current-clamp mode during patch-clamp experiments. Cells were either analyzed under control conditions (ctrl, left bars), or in the presence of 30 nM iberiotoxin (+IBTX, right bars). n (cells) = 9 for WT ctrl and WT + IBTX, 8 for BK-KO ctrl, 10 for BK-KO +IBTX. *p≤0.05, #p≤0.05 compared to respective WT condition. Statistical analysis was performed using Kruskal-Wallis test followed by Dunn’s multiple comparison test. (D) Basal FRET-ratio values ± SEM of MMTV-PyMT WT (left panel) and MMTV-PyMT BK-KO cells (right panel) expressing NES lc-LysM GEPII 1.0, a cytosolic K⁺ sensor. Experiments were either performed under control conditions (ctrl) or in the presence of 30 nM iberiotoxin (+IBTX). N (independent experiments) / n (cells analyzed) = 4/62 WT ctrl, 4/50 WT+IBTX, 4/35 BK-KO ctrl, 4/36 BK-KO +IBTX. *p≤0.05. ‡p≤0.001 compared to respective WT condition, Unpaired t-test (WT) or Mann-Whitney test (BK-KO and WT ctrl vs. BK-KO ctrl). (E) Representative images of MMTV-PyMT WT and MMTV-PyMT BK-KO cells, either under control conditions (left panel) or in the presence of 30 nM iberiotoxin (+IBTX, right panel). Brightfield images (top row), cyan fluorescence (second row), FRET (third row) and pseudocolored FRET-ratio images (fourth row) are demonstrated. (F, G) Representative whole-cell patch-clamp traces of MDA-MB-453 cells (F) and MCF-7 cells (G), 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. (H) 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 (third column) and a zoom (fourth column) 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 shown in Figure 1G. n (cells) = 18 for mtRFP and 16 for RFP-GPI. ***p≤0.001 Unpaired t-test. (I) Representative images of MCF-7 cells either expressing BK_Ca^RFP (left image) or BK_Ca-DEC^RFP (right image) additionally stained with MitoGREEN to visualize mitochondria. Images represent larger versions of the exact same images shown in Figure 1G. (J, K) Representative whole-cell patch-clamp traces of MCF-7 cells either expressing BK_Ca^RFP (J) or BK_Ca-DEC^RFP (K) either under control conditions (ctrl, left panels), or in the presence of 5 µM paxilline (+PAX, middle panels) or 30 nM iberiotoxin (+IBTX, right panels). (L) Global cellular RFP-fluorescence intensities of MCF-7 cells used for patch-clamp experiments. Cells either expressed BK_Ca^RFP (red bar) or BK_Ca-DEC^RFP (green bar). Data represents average ± SEM of 18 cells for both conditions.