Fig. 5
TSPO protects GB cells from TRAIL-induced apoptosis. a Resazurin assay to analyze the impact of TNFα, IFNγ, and FasL treatment on the viability of TSPO ± BTIC13 cells. Cells were treated for 48 h and fluorescence was normalized to untreated condition for control or TSPO shRNA-transduced cells. b Western blot analysis of total/cleaved caspase-9, total caspase-3, and total/cleaved PARP1 in TSPO ± BTIC13 cells upon 1, 3, and 6 h treatment with 50 ng/ml TNFα, IFNγ, and FasL (cl = cleaved; n.d. = not detected). c Resazurin assay to analyze the impact of TRAIL treatment on the viability of TSPO ± BTIC13 cells. Dose–response curves indicate normalized %-cell viability versus log-transformed 50 ng/ml TRAIL concentration. EC50 values were determined by nonlinear curve fitting. d Western blot analysis of total/cleaved caspase-9/-3 and PARP1 in TSPO ± BTIC13 cells upon 1 and 3 h treatment with TRAIL. e, f Resazurin assay to analyze the impact of (e) 50 ng/ml TNFα, IFNγ, and FasL and (f) TRAIL treatment on the viability of TSPO ± BTIC13 clones. g Luciferase-based caspase-3/-7 assay to measure caspase-3/-7 activation in TSPO ± BTIC13 clones after 4 h treatment with 50 ng/ml TRAIL. h Real-time cytotoxicity assay to analyze TRAIL-induced caspase-3/-7 activation over 24 h in BTIC13 clones. The graphs show the total apoptotic tumor cell area (green object area) per well. i, j Western blot analysis of total/cleaved caspase-9/-3 and PARP1 in TSPO ± (i) BTIC13 clones upon 1 and 3 h treatment with 50 ng/ml TRAIL and (j) BTIC129 cells upon 6 h treatment with 50 ng/ml TNFα, FasL and TRAIL. k Luciferase-based caspase-3/-7 assay to measure caspase-3/-7 activation in TSPO ± BTIC129 cells after 4 h treatment with TRAIL. l Real-time cytotoxicity assay to analyze TRAIL-induced caspase-3/-7 activation over 24 h in BTIC129 cells. The graphs show the total apoptotic tumor cell area (green object area) per well. Representative data of at least two independent experiments. e, f Cumulative data of three independent experiments. Values represent the mean of triplicates ± SD. P-value was calculated using two-tailed Student`s t-test (* = P < 0.05, ** = P < 0.01, *** = P < 0.005, **** = P < 0.001)