Fig. 2: GBM cells display augmented apoptotic response upon chaetocin and TRAIL combinatorial treatment.

a Caspase-3/7 activity analyses of U87MG cells upon chaetocin (100 nM) and TRAIL (100 ng/ml) combinatorial treatment. Data were normalized to untreated control cells. b Western blot analyses of U87MG cells for cleaved Casp8, Bid, t-Bid, Casp3 and PARP after pretreatment with chaetocin (100 nM for 24 h) followed by 6 h TRAIL (100 ng/ml) treatment. α-tubulin was shown as protein loading control. c Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay on U87MG cells showing increased DNA fragmentation upon chaetocin (100 nM) and TRAIL (100 ng/ml) combinatorial treatment for 24 h. Blue: DAPI staining nuclei, Green: TUNEL (+) cells. Scale bar: 100 µm. d Quantification of TUNEL staining by ImageJ program through counting TUNEL (+) cells with green fluorescence. e YO-PRO-1 and PI staining upon chaetocin (100 nM) and TRAIL (100 ng/ml) combinatorial treatment of U87MG cells for 24 h. Green: YO-PRO-1 staining apoptotic cells, Red: PI staining dead/necrotic cells. Scale bar: 200 µm. f Quantification of YO-PRO-1/PI staining by ImageJ program through counting green and red fluorescence positive cells. g Flow cytometric analysis of Annexin V/PI stained U87MG cells upon chaetocin (100 nM) and TRAIL (100 ng/ml) combinatorial treatment for 24 h. h Quantification of flow cytometry data showing marked increase in apoptotic cell populations upon combinatorial treatment. Data were normalized to total number of cells under each condition. i Cell viability analysis U87MG cells pretreated with caspase inhibitors (20 µM for 24 h) followed by chaetocin (100 nM) and TRAIL (100 ng/ml) treatment for 24 h in presence of inhibitors. Z-FA-FMK: negative control, Z-VAD-FMK: general caspase inhibitor. j Western blot analyses of U87MG cells showing individual stable CRISPR knockouts of DR5, BID, and Casp8, as well as double knock out Casp3/7 genes. GFP targeting guide RNA (g-GFP) was used as negative control for CRISPR assay. α-tubulin was shown as protein loading control. k Viability analysis of CRISPR edited U87MG cells upon combinatorial treatment with chaetocin (100 nM) and TRAIL (100 ng/ml) for 24 h. Data were normalized to untreated control. ((*) and (***) denote P < 0.05 and P < 0.001, respectively, two-tailed Student’s t-test).