Figure 7.

Evaluation of the susceptibility of MHT−treated U87 spheroids to NK cell−mediated killing (a–c). MHT−treated or untreated U87 cells were cultured for 24 h in serum-free neuronal stem cell medium; following this resting period, IL−2−activated NK cells were added to the culture. At day 7 each culture well was analyzed measuring the area of the spheroids using imageJ software. Representative images per each condition are reported (a). Results are presented as mean ± SEM of three experiments (b). Cell viability was also evaluated via crystal violet staining (c). U87 cells were gently harvested and incubated in conventional adhesion conditions. After 48 h, cell cultures were stained with crystal violet followed by extensive washes (to remove non-adherent cells) and lysis of adherent cells. Relative absorbance at 595 nm was measured with a plate reader. Data are expressed as mean ± SD of three experiments. Chemotaxis of NK cells toward chemokines produced by MHT−treated cells was evaluated using a transwell migration assay (d). Conditioned medium collected from U87 cells untreated or treated with IONCs and MHT were added to the lower chambers of transwell plates. Fresh medium was used as a control. NK cells (1 × 10⁵) were added to the upper chamber and cells were incubated for 18 h. Cells in the lower chambers were harvested and counted. Data are shown as mean ± SEM of four independent experiments. Statistical analysis was performed using a one−way ANOVA test (* 0.01 < p < 0.05; ** 0.001 < p < 0.01; *** p < 0.001; n.s. = not significant).