Fig. 2. TWIST1 KO reduces the neurosphere forming capacities of NB cells in vitro and the tumor growth capacities of SK-N-Be2c cells in vivo.

a Representative images (scale bar 200 µm) showing the size and shape of primary neurospheres of Control and sgTWIST1 NB cells after 7 days in culture. The numbers of cells obtained after dissociation of Control and sgTWIST1 primary neurospheres are plotted in bar graphs as individual values for each independent experiments and mean ± SD (n = 5 experiments performed in duplicates). Mann−Whitney test: *p = 0.0317 for SK-N-Be2c; *p = 0.0159 for LAN-1 and NB1-M. b Kaplan−Meier survival curves of athymic Swiss nude mice implanted orthotopically with SK-N-Be2C-Control or -sgTWIST1 cells. Mice were sacrificed once tumors reached the volume of 1000 and 500 mm³ for ortho_1 and ortho_2 experiments, respectively. Tumor take: ortho_1: 100% (6/6) in the Control group, 66.66% (4/6) in the sgTWIST1 group; ortho_2: 89% (8/9) in the Control group, 83% (10/12) in the sgTWIST1 group. Median survival in the Control vs sgTWIST1 group: 26 vs 44 days for ortho_1 (**p = 0.0027); 49 vs 78 days for ortho_2 (**p = 0.0016). Gehan−Breslow−Wilcoxon test. c Tumor growth (mean tumor volumes ± SD) for ortho_1 experiment. Multiple t-test (HolmSidak, α = 0.05, without assuming a consistent SD): **p = 0.0037. d Time for tumor initiation and tumor growth in the ortho_2 experiment (individual values for each mice and mean ± SD). Tumor initiation corresponds to the number of days required to measure an AG volume > 10 mm³ (mean Control: 41.38 days, sgTWIST1: 64.10 days, *p = 0.0192). Time for tumor growth was calculated as the number of days at sacrifice minus the number of days for tumor initiation (mean Control: 9.25 days, sgTWIST1: 22.50 days, ***p = 0.0006, unpaired t-test).