Extended Data Fig. 8: Tumor biological effects of KCa3.1 overexpression, and further in vitro characterizations.

a, KCa3.1 overexpression (OE) versus control; S24 line. b, KCa3.1 overexpression (OE) versus control; P3 line. a1 and b1, Relative expression of KCa3.1 as determined via qPCR; n = 3 technical replicates. a2, Normalized KCa3.1 immunofluorescence values of all cells (see Methods for more details). a3, Whole-cell patch-clamp electrophysiology of S24 cells overexpressing KCa3.1; arrows indicate glioblastoma cells connected via gap junctions filled via the patch pipette containing Alexa 594 dye; on the right side, I-V curves of voltage ramps between −105 mV to 55 mV are shown before (grey trace) and after wash-in of 1 µM TRAM-34 (red trace). TRAM-34 sensitive currents were detected in all S24 KCa3.1 overexpression cells (n = 4 cells), but in none of the control S24 cells (n = 3 cells). b2, Fraction of periodic cells of all active cells in vitro. a4, and b3, Frequency of Ca²⁺ oscillation of periodic cells in vitro. a5 and b4, Global Ca²⁺ activity in vitro. a6, Ca²⁺ traces of each cell from representative recordings; dark red: periodic cells. a7 and b5, Fraction of EdU-positive cells in vitro. a8, and b6, Fraction of dead cells in vitro. a2, n = 1810 cells (S24 OE control) and n = 1532 cells (S24 KCa3.1 OE) from 6 FOVs, respectively; a4–5, and b2–4, n = 10 recordings (S24 OE control), n = 11 recordings (S24 KCa3.1 OE), n = 6 recordings (P3 OE control), n = 7 recordings (P3 KCa3.1 OE) from 2 biologically independent experiments per group, respectively. a7–8, and b5–6, n = 27 FOVs from 2 biologically independent experiments per group, respectively. a1, a4–8, b1–2, and b4, two-sided t-test. a2,b3, and b5–6, two-sided Mann-Whitney test. c, Representative Nestin and KCa3.1 double-immunostaining after Ca²⁺ recordings of the same region in vitro; arrows indicate cells that displayed periodic Ca²⁺ oscillations in the preceding Ca²⁺ recording; S24 line. d, Normalized KCa3.1 immunofluorescence values after calcium recordings in periodic cells and non-periodic cells (see Methods for more details); periodic cells show a 51.1% ± 11.1% (95% CI) for S24 and 43.2% ± 17.0% (95% CI) for BG5 higher fluorescence intensity than non-periodic cells. c,d, n = 3177 S24 non-periodic cells, n = 75 S24 periodic cells, n = 3108 BG5 non-periodic cells, and n = 29 BG5 periodic cells from in n = 5 recordings per cell line from n = 3 biologically independent experiments; Kruskal-Wallis test, Dunn’s test. e, fraction of KCa3.1^high tumor cells growing in mice; n = 27 regions in 4 mice (S24 line) and n = 21 regions in 3 mice (BG5 line). f, Fraction of tumor cells with the respective number of TMs per tumor cell in KCa3.1^high and KCa3.1^low cells; immunostainings of tumors growing in mice; error bars show s.d.; S24 line; n = 12 regions in 3 mice; Kruskal-Wallis test, Dunn’s test. g, Representative KCa3.1 and EdU immunostainings of S24 line; arrows indicate the KCa3.1^high cells; dashed circles indicate the radius of 100 µm around the KCa3.1^high cells; n = 18 recordings from 2 biologically independent experiments. h–k, Fraction of EdU-positive cells with a distance < 100 µm versus > 100 µm to the closest KCa3.1^high cell normalized to the fraction of EdU-positive cells of each recording; h, S24 line control conditions, n = 106 ROIs (<100 µm) and n = 30 (>100 µm) in 2 biologically independent experiments, i, BG5 line control conditions, n = 36 ROIs (<100 µm) and n = 12 (>100 µm) in 2 biologically independent experiments, j, S24 line BAPTA-AM treatment, n = 42 ROIs (< 100 µm) and n = 11 (>100 µm) in 2 biologically independent experiments, and k, BG5 line BAPTA-AM treatment, n = 31 ROIs (<100 µm) and n = 12 (>100 µm) in 2 biologically independent experiments; two-sided Mann-Whitney test. Error bars show s.e.m. ns, not significant (P ≥ 0.05).

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