Fig 2: Role of oxidative stress in overcoming Temozolomide resistance. (A) Viability analysis and (B) gene expression profile for apoptotic-related genes (BAX, BAD and BCL-2) in U251 and (C,D) in T98 cells. Viability was assessed by means of Trypan blue exclusion test after treatment with 200µM or 1mM H2O2 ± 100 µM Temozolomide (TMZ). Data of viability were expressed as percentage of viable cells; ** p < 0.01; *** p < 0.001 treated vs. control cells. (E) Gene expression profile for CMA-related genes (LAMP2A, HSC70, PHLPP1), and for (F) HIF-1a and VEGF in U251 and in (G,H) T98 cell after treatment with 200 µM or 1 mM H2O2 ± 100 µM TMZ. * p < 0.05; ** p < 0.01; *** p < 0.001 treated vs. control cells. All data of gene expression were normalized for ß-ACTIN, and the ??Ct values were expressed as FOI of the ratio between treated and control cells. * p < 0.05; ** p < 0.01; *** p < 0.001 treated vs. control cells. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. TMZ-treated cells. (I) Scratch test and (L) gene expression for EMT-related genes (SLUG, SNAIL, E-CAD) performed after treatments in U251 and (M,N) T98 cells. Wound closure percentage compared to control was analyzed with Image J software. ** p < 0.01 treated vs. control cells. ## p < 0.01 vs. TMZ-treated cells. Data of gene expression were normalized and expressed as mentioned above. * p < 0.05; ** p < 0.01; *** p < 0.001 treated vs. control cells. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. TMZ-treated cells.

Fig 3: Essential chaperone-mediated-autophagy related gene involvement in Temozolomide response in U251 and T98 cells. (A) Western blot analysis of LAMP2A, HSC70 and GAPDH protein levels in subcellular fractions (Cyto = cytosolic fraction; Lyso = Lysosomal fraction) obtained from untreated (-) and Temozolomide (TMZ) treated (+) U251 and T98 cell lines. (B) Protein signals were normalized for the respective fraction marker: TUBULIN (cytosol) and LAMP1 (lysosomes). Histograms show the distribution of target proteins in the fractions compared to untreated control levels. ** p < 0.01 treated vs. control cells. Lysosomal content of the three considered proteins are significantly different in U251 compared to T98 cells (LAMP2A p = 0.003; HSC70 p = 0.04; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) p = 0.03). (C) Viability analysis and (D) assessment of HIF-1a activity in U251 and in (E,F) T98 cells after PHLPP1 or HSC70 silencing ± 100 µM TMZ treatment. Data were expressed as percentage of viable cells and as RLU, as previously described. ** p < 0.01 treated vs. control cells; ### p < 0.001 vs. TMZ-treated cells. (G) Gene expression profile for CMA-related genes (LAMP2A, HSC70, PHLPP1) in U251 and (H) T98 cells. (I) Gene expression profile for EMT-related genes (SLUG, SNAIL, E-CAD) in U251 and (L) T98 cells. Gene expression was analyzed by means of Real-time PCR. All data were normalized for ß-ACTIN, and the ??Ct values were expressed as FOI of the ratio between treated and control cells. * p < 0.05; ** p < 0.01; *** p < 0.001 treated vs. control cells; ## p < 0.01, ### p < 0.001, vs. TMZ-treated cells. Mean values ± SD of three independent experiments.