FIGURE 6 from Activation of the Mevalonate Pathway in Response to Anti-cancer Treatments Drives Glioblastoma Recurrences Through Activation of Rac-1

Treatment-induced upregulation of the mevalonate pathway in glioma affects stemness through prenylation of Rac1. A, HK374 cells were treated with radiation in the presence or absence of QTP (10 µmol/L) and/or atorvastatin (1 µmol/L) for 48 hours. The activated Rac1 was immunoprecipitated by 10 µg PAK-PBD agarose beads from the whole cell lysates and subjected to immunoblotting against Rac1, along with the total proteins. His-tagged Rac1 protein serves as the positive control. B, The densitometry measurements of activated Rac1/total Rac1 using Image J. C, Transwell migration assay of HK374 cells pretreated with radiation in the presence or absence of QTP (10 µmol/L) and/or atorvastatin (1 µmol/L) for 48 hours. D, The quantification of migrated cells using Image J. E, Confocal images of microtubules in HK374 cells treated with radiation in the presence or absence of QTP (10 µmol/L) and/or atorvastatin (1 µmol/L) for 48 hours. White arrowheads: filopodia. Yellow arrowheads: TNTs. F and G, Rac1 knockdown efficiency was evaluated at both mRNA (qRT-PCR) and protein (Western blotting) levels at day 2 and day 7 after siRNA transfection. β-actin was used as the loading control and the densitometry measurements of Rac1 were performed using Image J. H and I, Clonogenic assay of siCtrl or siRac1 transfected HK374 cells treated with radiation in the presence or absence of QTP (10 µmol/L) and/or atorvastatin (1 µmol/L) for 7 days and the resulting plating efficiencies. J, Sphere-forming capacity of siCtrl or siRac1 HK374 spheres treated with radiation in the presence or absence of QTP (10 µmol/L) and/or atorvastatin (1 µmol/L). K, log fraction plot generated by extreme limiting dilution assay (ELDA), where the y axis “log fraction nonresponding” indicates frequency of cells incapable of forming clonal spheres and the x axis “dose (number of cells)” indicates number of cells per mL. The slope of the line is the log-active cell fraction, and dotted lines give the 95% confidence interval. All experiments have been performed with at least three biological independent repeats. P-values were calculated using one-way ANOVA for B and D, unpaired Student t tests for F and G; two-way ANOVA for I and J. ns: not significant.