Ttyh1 knockdown alters TM phenotypes in vivo. a, Verification of shRNA knockdown of Ttyh1 in S24 (left) and T269 (right) GBMSCs by Western blot analysis (30% reduction in protein level in S24; 96% reduction in T269). b, AlamarBlue viability and proliferation assay of S24 shControl and shTtyh1 cells demonstrates that the metabolic activity/viability [as measured on day 0 (D0)], as well as the proliferation (exponential growth phase shown), is not significantly altered by the shRNA-mediated knockdown (n = 12 replicates for each shControl and shTtyh1; ANOVA on the ranks; mean ± SEM). c, d, Exemplary flow cytometry analyses of S24 shControl and shTtyh1 revealed no significant differences in the Ki67⁺ growth fraction (c; n = 3 replicates for shControl and shTtyh1 respectively; 2-sided t test; p = 0.066) or apoptosis rate (d; n = 3 replicates for shControl and shTtyh1 respectively; 2-tailed t test; p = 0.289). Red numbers indicate populations in percentage. e, f, Exemplary in vivo 3D MPLSM images of S24 shControl and shTtyh1 GBMSC TMs (e) as well as T269 shControl and shTtyh1 GBMSC TMs (f). Note that shTtyh1 cells show morphologically altered TMs with filament beading (arrowheads). g, Observations of filament beading by MPLSM in vivo itemized regarding cellular subpopulations (n = 6 mice, S24, day 20; n = 5 mice, T269, day 21 ± 2; Mann–Whitney tests). h, Exemplary in vivo MPLSM images of TM-rich multipolar tumor cells in S24 shControl and S24 shTtyh1 tumors showing no phenotypic changes in this cellular subtype. i, Relative fractions of GBMSCs with zero, one or two, and >4 TMs in S24 shControl versus shTtyh1 tumors (20–25 d after implantation; n = 3 mice per group; 2-tailed t tests). j, Absolute number of tumor cells per mm³ categorized regarding these cellular subtypes (n = 3 mice per group; ANOVA on ranks). k, Quantification of the fractions of TM-connected GBMSCs in the cellular subgroups with 1–2 TMs versus >4 TMs, demonstrating that most cells with >4 TMs are connected, whereas the cells with 1–2 TMs are mainly unconnected to other tumor cells in this glioma via their TMs (S24 shControl: day 20 ± 5; S24 shTtyh1: day 20; n = 4 mice per group; 1-way ANOVA). e–k: data obtained by in vivo MPLSM. *p < 0.05; ***p < 0.001.