Figure 2
Treatment with aporphine and isoquinoline derivatives induce an intrinsic apoptosis cell death. (A) COX activities of GBM cells after 24 h of incubation with A5 (30 μM) or C1 (30 μM). Results are expressed as percentage of cell control treated with DMSO. (B) Representative immunoblot of cleaved caspase-3 in U3017MG cells treated with TMZ, APO, A5 or C1 for 48 h. β-Actin was used as a loading control, and molecular mass (kDa) markers are indicated along with densitometric values of normalized band intensity. (C, D) RT-qPCR was used for detection of markers for cell death mediated by apoptosis: BIM, BECN1 and BAX in U3017MG (C) and U3031MG (D) cells treated with DMSO, TMZ (150 µM), APO (25 µM), A5 (30 µM) or C1 (30 µM) for 48 h. TMZ, an alkylating agent frequently used as chemotherapy in GBM patients, was used as the positive control. (E, F) RT-qPCR was used for detection of DNA damage response gene expression: CDKN1A (p21) in U3017MG (E) and U3031MG (F) cells treated with DMSO, TMZ (150 µM), APO (25 µM), A5 (30 µM) or C1 (30 µM) for 48 h. All the relative expression levels were normalized to GAPDH expression and calculated using the 2−ΔΔCt method. Error bars represent SD from three biological replicates and p values were calculated according to the two-way ANOVA test followed by Bonferroni (post-test); *p < 0.05, **p < 0.01, ***p < 0.001.
