Extended Data Figure 6: Prolonged currents in pediatric glioma amplified by gap-junction coupling.
a, Input resistance of non-responding and depolarizing cells (n=29 fast EPSC, n=247 prolonged current, n=319 no response). b, Biocytin dye filling illustrates coupling of xenografted glioma cells that exhibit prolonged currents (n=7/2 slices/mice); Red = streptavidin-biocytin; green = GFP. Scale bar, 100μm. c, Prolonged currents in glioma largely blocked by carbenoxolone (CBX, 100μM). Red box highlights zoomed in view of representative trace to illustrate residual slow current after application of CBX. d, Time course of prolonged current in glioma cells (black) and pyramidal cell EPSC (blue) responses to addition of CBX with subsequent washout of inhibitor (duration=red bar; n=6/6 glioma cells/mice; n=5/2, neurons/mice). e, Bar graph with additional data quantifies the drop in current amplitude after addition of CBX (n=19/11 cells/mice; data also shown in Fig.3l). f, Input resistance of cells in (d) in response to CBX. g, Quantification of (f) (n=19/11 cells/mice). h, Time course of prolonged current in glioma in response to addition of meclofenamate (100μM; duration=red bar; n=8/3 cells/mice). i, Quantification of (h); data also shown in Fig.3l; left. Representative traces illustrating block of prolonged current amplitude after addition of meclofenamate (red, right). j, Input resistance of cells in (h) in response to addition of meclofenamate (n=8/3 cells/mice). k, Quantification of (j). l, Representative frames from two photon calcium imaging of in situ SU-DIPGXIII-FL glioma xenografts illustrating spontaneous transients before (top) or after addition of CBX (bottom). Red = tdTomato nuclear tag in glioma cells; green = GCaMP6s. Scale bar, 50μm; Results replicated across 3 mice. m, Synchronicity analyses of spontaneous calcium transients before (top) or after addition of CBX (bottom). n, Representative trace of GCaMP6s peak intensity over time (s) in a single cell before (black) and after addition of CBX (red). Data plotted as ρF/Fo; n=3 mice. o, Synchronicity scores of individual cells within glioma xenograft before and after addition of CBX (n=164 cells across 3 mice). y-axis represents synchronicity score, x-axis represents number of cells with specific score. For a, d, e, f, g, h, i, j, and k, data are shown as means ± s.e.m. P-values determined by one-way ANOVA for (a), and by one-tailed Wilcoxon matched-pairs signed rank test. for (e,g,i,k,o). **P<0.01. ****P<0.0001. NS indicates P > 0.05.