Fig. 5

Co-treatment of Birinapant with TRAIL can synergistically increase their efficiency and induce apoptosis in colorectal adenocarcinoma cells in 2D and 3D. 5a: Cell viability of cell line HT29 after co-treatment with the SMAC-mimetic Birinapant and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with Birinapant, TRAIL and their combination for 48 and 72 h and the % percentage cell viability was measured. The average of three independent experiments is presented. Columns = % percentage of cell viability, bars = SD. 5a-RKO: Respectively for cell line RKO (upper panel). To check for the reversibility of treatment effects, RKO cells were either left untreated (ctr = control), treated with either Birinapant, TRAIL or their combination for 48 h. Cells were then incubated with additional 48 h without treatments (NoT) and the % percentage cell viability was measured. The average of three independent experiments is presented. 5b-I. Protein levels of cIAP-1 and XIAP after 0.5, 1 and 5 μM AT-406 (lanes 3–5) or Birinapant (lanes 6–8) treatments respectively for 48 h, compared to control lanes 1 (no treatment) or 2 (DMSO treatment). 5b-II and 5b-III. Protein levels of PARP-1 and total Caspase-3 in HT29 cell line after co-treatment with SMAC-mimetic Birinapant and TRAIL for 48 and 72 h. Cells were either left untreated (lane 1) or treated with DMSO (lane 2), with 5 μM Birinapant (lane 3), with 10, 50 and 100 ng/mL TRAIL (lanes 4–6), or with 5 μM Birinapant and combination with TRAIL (lanes 7–9) for 48 and 72 h. Sensitive cell line HCT116 treated with TRAIL is used as positive control (lane 10). Using W. B., protein levels of PARP-1 (Figure 5b-II) and of total Caspase 3 (Figure 5b-III) were analyzed after the corresponding treatments. Data are representative for three independent experiments. 5b-I-RKO: Protein levels of cIAP-1 and XIAP after mono-treatments (lanes 3 and 4 for AT-406 and Birinapant respectively and lanes 5–7 for TRAIL 10, 50 and 100 ng/mL respectively) and co-treatments (lanes 8–13 as shown) for 24 h. Untreated (lane 1) and treated with DMSO (lane 2), cells are also presented. 5b-II-RKO and 5b-III-RKO: Protein levels of PARP-1 and total Caspase-3 after respective mono-treatments and co-treatments (same lines as 5-I-RKO). Data are representative of three independent experiments. 5C: Light microscopy images from HT29 culture after combined treatment with Birinapant and TRAIL. Detached (apoptotic) cells are shown in supernatant of co-treated groups for 48 and 72 h. Representative images. 5d: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combination. Nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images.. Scale bar: 9.9 μm. 5e. Light microscopy of three-dimensional culture of HT29 cells after co-treatment with Birinapant, TRAIL and their combinations in 3D for 6 days. Representative images. 5f: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combinations in 3D culture for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 57.4 μm