Extended Data Fig. 6: Genetic perturbation of KCa3.1 reduces intrinsic rhythmicity, global Ca²⁺ activity, and tumor cell proliferation.

a, KCa3.1 knockout (KO; 2 different sgRNAs) versus knockout control (non-targeting sgRNA); S24 line. For both KOs no KCa3.1 signal was detected. Therefore, the qPCR cycle limit +1 was used as the hypothetical timepoint of detection for quantifying the relative KO of KCa3.1. b, KCa3.1 knockdown (KD) versus knockdown control (non-targeting shRNA); S24 line. c, KCa3.1 knockdown (KD) versus knockdown control (non-targeting shRNA); P3 line. a1–3,b1, and c1, Relative expression of KCa3.1 as determined via qPCR; n = 2 technical replicates (S24 KO control, S24 KCa3.1 KO-1 & −2), n = 5 technical replicates from 2 biologically independent experiments (S24 KD control, S24 KCa3.1 KD), n = 3 technical replicates (P3 KD control, P3 KCa3.1 KD). a4 b2, and c2, Fractions of periodic cells of all active cells in vitro. a5,b3, and c3, Frequencies of Ca²⁺ oscillations of periodic cells in vitro. Note that the difference between the groups in b3 reaches statistical significance but is minor, and thus of questionable biological relevance. a6,b4, and c4, Global Ca²⁺ activity in vitro. a7, Ca²⁺ traces of each cell from representative recordings; dark red: periodic cells. a8,b5, and c5, Fraction of EdU-positive cells in vitro. a9,b6, and c6, Fractions of dead cells in vitro. a4–6,b2–4, and c2–4, n = 5 recordings (S24 KO control, S24 KCa3.1 KO-1 & −2), n = 6 recordings (S24 KD control and S24 KCa3.1 KD), n = 5 recordings (P3 KD control and P3 KCa3.1 KD) from 2 biologically independent experiments per group, respectively. a8-9,b5–6, and c5–6, n = 27 FOVs (S24 KO control (EdU) and S24 KCa3.1 KO-1 & −2 (EdU)), n = 18 FOVs (S24 KO control (dead cells) and S24 KCa3.1 KO-1 & −2 (dead cells)), n = 45 FOVs (S24 KD control (EdU) and S24 KCa3.1 KD (EdU)), n = 18 FOVs (S24 KD control (dead cells) and S24 KCa3.1 KD (dead cells)), n = 27 FOVs (P3 KD control and P3 KCa3.1 KD) from 2 biologically independent experiments per group, respectively. a4–6,a8, One-way ANOVA, Dunnett’s test. a9, Kruskal-Wallis test, Dunn’s test. b1–6,c1–2, and c4, two-sided t-test. c3, and c5–6, two-sided Mann-Whitney test. d, Representative immunofluorescence images, e, tumor cell density f, fraction of Ki67-positive tumor cells, and g, fraction of activated caspase-3-positive tumor cells of tumor-bearing cerebral human organoids after implantation of S24 KO control and S24 KCa3.1 KO-2 line; n = 12 regions in 6 human cerebral organoids per group (e,f) and n = 6 regions in 3 human cerebral organoids per group (g); two-sided t-test (e,g) and two-sided Mann-Whitney test (f). Error bars show s.e.m. ns, not significant (P ≥ 0.05).

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