Fig. 3: RALB silencing regulates DR5 expression levels.

A Top Left: HCT116 cells were transfected with 10 nM SC, siRALA, siRALB or siRALGDS for 24 h, following which cells were harvested for protein extraction. Protein was subsequently analysed using a human apoptosis array. Densitometry was performed on the array panels using ImageJ software, and results for DR4, DR5, Fas/CD95, FADD and TNFR1/TNFRSF1A are shown. Top right: PARP, DR5, DR4, FADD, XIAP, Bcl-xL, RALA and RALB levels in CRC cells following RALA, RALB and RALGDS silencing for the indicated times. Bottom left: Wheat germ agglutinin (WGA), a lectin that binds N-acetylglucosamine post-translational modifications on membrane receptors, was used as a plasma membrane marker. Fixed cells were stained with Alexa488-WGA prior to permeabilisation to minimise the staining of glycosylated proteins in the Golgi apparatus. A single confocal image was collected of the Alexa488-WGA stained membrane (green) alongside immunostained endogenous DR5 (red). Laser settings were kept constant between the samples to enable the comparison of membrane-associated DR5 staining in control (SC) and siRALB-treated cells. Bottom right: >20 cells were scored for intensity in ImageJ. The intensity was normalised to cellular area and plotted. The data are representative of three independent experiments. B CRC cells were transfected with 10 nM SC or siRALB for 24 h and DR5 cell-membrane expression was assessed by flow cytometry using a DR5-specific phycoerythrin-conjugated mAb. Expression was compared with an isotype-matched control antibody (IgG control). Geometric means (GM) for fluorescence intensity were plotted and significance was analysed using an unpaired t-test. C CRC cells were transfected with 10 nM siRNA targeting DR4 or DR5 for 24 h and were thereafter transfected with 10 nM RALB siRNA for 24 h. Apoptosis was assessed using WB for PARP, cleaved Caspase-8 and Caspase-3 (top) and Caspase-3/7 activity (bottom). (Asterisk next to the western blot indicates an unspecific band). D Paired CRISPR parental (control = CT) and DR5 knockout (KO) cells were transfected with 10 nM SC or RALB siRNA (siRB) for the indicated time. Apoptosis was determined by WB for PARP, Caspase-8 and Caspase-3 (left), Caspase-3/7 activity (right, top) and PI flow cytometry (right, bottom). E WB analysis for PARP and Caspase-8 (left) and Caspase-3/7 activity (right) of the effect of TRAIL neutralising antibody (nAb) treatment (100 ng/ml) on cell death in HCT116 cells transfected for 48 h with 10 nM SC or siRALB.