Fig. 5: RALB inhibition enhances TRAIL-induced apoptosis in KRASMT CRC.

A CRC cells were transfected with 10 nM RALB siRNA for 24 h prior to treatment with rhTRAIL for the indicated time, (CT = control, untreated sample). Apoptosis was determined by using WB analysis for PARP, cleaved Caspase-8, -9 and -3 levels (top) and Caspase-3/7 activity (bottom). LE = long exposure. B HCT116 and SW620 cells were transfected with either SC or siRALB for 24 h followed by rhTRAIL treatment for a further 24 h. Apoptosis was assessed using Annexin V/propidium iodide (PI) staining by high-content screening. The graph indicates the percentage of positive stained cells. C HCT116 and SW620 CRC cells were transfected with siRALB or control siRNAs and then treated with the indicated doses of rhTRAIL for 72 h. An MTT assay was used to evaluate cell viability, which is presented relative to SC control on the graphs. The dashed line indicates a 50% change in cell viability. D HCT116 cells were treated with Dinaciclib for 24 h, prior to treatment with rhTRAIL for 24 h. WB analysis for PARP, cleaved Caspase-8 and -3 (Left top), Caspase-3/7 activity assays (left bottom) and PI flow cytometry analysis of the sub-G₁ apoptotic population (right bottom). HCT116 cells were treated with Dinaciclib for 48 h and expression of active (GTP-bound) and total RALB was evaluated using WB. CT refers to an untreated sample (right top).