Figure 6

HDAC inhibitors are anti‐invasive and selectively cytotoxic toward TSC2^−/− cells xenotransplanted into zebrafish. A) Representative phase contrast image of 1 day post‐injection (dpi) zebrafish larvae injected with TSC2^−/− mCherry⁺ cells into the hindbrain ventricle (hbv). B) Representative images of zebrafish larvae injected with mCherry⁺ WT or TSC2^−/− cells into the hbv. Fish were imaged 1 and 4 dpi. Scale bars of 200 µm. C,D) Quantification of cell invasion using automated invasion analysis. Images analyzed in (D) were taken 4 dpi following 3 day treatment ± 20 × 10⁻⁹m rapamycin (mean ± SD; * = p < 0.05 by the Kruskal–Wallis test with Dunn's post hoc comparisons; n = 38–73). E) Number of mCherry⁺ cells detected per zebrafish following whole larvae dissociation at 4 dpi and analysis by flow cytometry. Samples were treated for 3 days ± 20 × 10⁻⁹m rapamycin. Each replicate is a pool of 15–20 zebrafish larvae (mean ± SD; * = p < 0.05 by one‐way ANOVA; n = 6). F) Percentage of CASP3⁺ cells in the mCherry⁺ population from whole larvae dissociation at 4 dpi, following 3 day treatment ± 20 × 10⁻⁹m rapamycin. Each replicate is a pool of 15–20 zebrafish larvae (mean ± SD; * = p < 0.05 by one‐way ANOVA; n = 5–6). G,H) Effect of 3 day HDAC inhibitor treatment (20 × 10⁻⁶m SAHA, 5 × 10⁻⁶m SB939, 1 × 10⁻⁶m LBH589) ± 20 × 10⁻⁹m rapamycin. G) Invasion scores calculated on images acquired 4 dpi (mean ± SD; * = p < 0.05 by the Kruskal–Wallis test with Dunn's post hoc comparison to vehicle‐treated; n = 27–73). H) Percentage of CASP3⁺ cells in the mCherry⁺ population from whole larvae dissociation at 4 dpi. Each replicate is a pool of 15–20 zebrafish larvae (mean ± SD; * = p < 0.05 by ANOVA with Dunnett post hoc comparison to vehicle‐treated; n = 3–6). Not all outliers in (C,D) and (G) are visualized due to trimmed axes (although outliers were included in mean ± SD and the statistical calculation).