Fig. 4

Pre-treatment with Birinapant and then co-treatment with Birinapant/ BRAF inhibitor PLX4720 synergistically induce apoptosis of colorectal adenocarcinoma cells in 2D and 3D. a: Cell viability after co-treatment with the SMAC-mimetics Birinapant or AT-406 in combination with the BRAF inhibitor PLX4720. Cells were either left untreated (ctr = control) or treated with 5 μM Birinapant, 5 μM AT-406 and 1 μM PLX4720 and their combinations for 48 and 72 h. The average of three independent experiments is presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. b: Cell viability after pre-treatment with the SMAC-mimetic Birinapant and then co-treatment with BRAF inhibitor PLX4720 and Birinapant. Cells were either left untreated (ctr = control) or treated with 0.5 or 1 μM Birinapant and 0.2 or 0.5 μM PLX4720. For the pre-treatment testing, cells were first incubated for 24 h with either 0.5 or 1 μM of Birinapant and then co-treated with 0.2 and 0.5 μM PLX4720 for another 24 or 48 h. c: Protein levels of PARP-1, total Caspase-3, XIAP, cIAP-1 and p-ERK1/2 in RKO by W.B., after pre-treatment with 0.5 or 1 μM Birinapant and then co-treatment with 0.2 or 0.5 μM PLX4720. Untreated cells were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. d: Light microscopy images from RKO culture after pre-treatment with Birinapant and then co-treatment with PLX4720. Detached cells are shown in supernatant of co-treated group. Several images were taken from untreated and treated with Birinapant (0.5 or 1 μM) and PLX4720 (0.2 or 0.5 μM) RKO cells while been cultured in 6-well plates for 48 and 72 h. Representative images are presented. e: Light microscopy of three-dimensional culture of RKO cells after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and their combination in 3D culture for 6 d. Representative images. f: Confocal microscope images were taken after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and combinations in 3D cultures for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 20 μm