Fig. 4

Effects of 2-DG and lovastatin on cell survival, cell dissemination, and radio- and chemotherapy. (A) Neutral red assay was performed to measure cell viability in control and myrAkt1 clones treated for 48 h with increasing 2-DG and lovastatin doses (n = 3). Data are mean ± SEM, ** p-value < 0.001; *** p-value < 0.0001; one-way Anova test. (B) Cells (1 × 10⁵) were seeded into 6-multiwell plates. After 24 h, cells were treated with 2 mM 2-DG and 10 µM lovastatin or DMSO vehicle. After 48 h, cells were stained with PI and Annexin V. The percentages of viable, early, and late apoptotic cells were calculated by FACS analysis and are reported in the graph (n = 2). (C) Gel zymogram depicting differences in MMP-2 and -9 expression in myrAkt1 cells treated with 2 mM 2-DG and 10 µM lovastatin or DMSO vehicle for 24 h (n = 2). (D) MyrAkt1 cells were pre-treated for 2 h with 2 mM 2-DG and 10 µM lovastatin or DMSO vehicle before wound induction. As depicted in the graph, the wound repair area was evaluated over a time-course of 24 h. Representative images were taken after 24 h (n = 2). Data are mean ± SEM, * p-value < 0.05; *** p-value < 0.0001; one-way Anova test. (E) CM-Dil fluorescent labeled myrAkt1 cells were pretreated with 2 mM 2-DG, 10 µM lovastatin or DMSO vehicle 24 h prior to yolk injection into zebrafish embryos. Xenografted embryos were maintained in water added with 50 µM 2-DG, 0.1 µM lovastatin, or the DMSO vehicle. Representative images were taken after 4 dpi at 20× and 32× magnification. Quantification of migrated tumor cells was calculated at 2 and 4 dpi (n = 3). Data are mean ± SEM, *** p-value < 0.0001; one-way Anova test. (F) Clonogenic and neutral red assays (left and right graphs, respectively) were performed to measure viability of cells preincubated for 2 h with 2 mM 2-DG, 10 µM lovastatin, or the DMSO vehicle before irradiation (4 Gy) and doxorubicin treatment (1 µM) (n = 3). Data are mean ± SEM, ** p-value < 0.001; *** p-value < 0.0001; one-way Anova test.