Fig. 4: DR5 expression is regulated by RALB through lysosomal degradation and p53-mediated transcription.

A Left: HCT116 cells were transfected with 10 nM of SC or 4 different siRNA sequences targeting RALB for 24 h. TNFRSF10B (upper) and RALB (lower) mRNA was quantified using RT-PCR. Raw values were normalised to the expression of housekeeping genes ACTB and GAPDH and were analysed using the ΔΔCT method. mRNA levels presented are relative to SC. Middle top: HCT116 p53 wild-type (WT) and null cells were transfected with either 10 nM SC or siRALB (two different sequences) for 24 h and the protein expression of p53, DR5 and RALB was determined using WB. Middle bottom: HCT116 cells were transfected with 10 nM of SC or four different siRNA sequences against RALB for 24 h. TP53 mRNA was quantified using RT-PCR. Analysis was performed as described above. Right: SW620, GP5d and LoVo cells were transfected with 10 nM SC or RALB siRNA (_6) for 24 h and DR5 expression was determined by WB. B Left: HCT116 cells were treated with siRNA targeting either SC or RALB for a total transfection time of 24 h. Cells were treated with 20 µM Chloroquine (CQ) for 4, 6 or 8 h. The expression of DR5, RALB, LC3-A/B and β-actin was analysed using WB. The numbers at the bottom of the WB panel represent densitometry performed on the DR5 blot using ImageJ. Middle: HCT116 cells were transfected with siRNA targeting either SC or siRALB and were treated with 50 nM Bafilomycin A1 for 3 h for a total transfection time of 24 h. The expression of DR5, RALB and β-actin was analysed using WB. The numbers at the bottom of the WB panel represent densitometry performed on the DR5 blot using ImageJ. Right: HCT116 cells were transfected with 1 µg of a WT FLAG-tagged RALB construct for 24 h followed by treatment with 20 µM Chloroquine for a further 24 h and DR5 and FLAG-tag expression was determined by WB. C Top: HCT116 cells were transfected with either empty vector (EV) or FLAG-RALB WT for 24 h. Cells were then fixed, permeabilised, blocked and stained with a lysosomal marker LAMP1, and DR5 antibody. Bottom left: Manders’ colocalisation coefficient (MCC) was calculated using ImageJ to measure the fraction of total DR5 fluorescence overlapping with LAMP1 fluorescence for each treatment and is presented in the ‘Merge’ image rounded to two decimal places. Image presented is representative of three independent experiments. Additional cells are presented in Fig. S5J. The MCC was calculated for over 50 cells across each treatment group over three independent experimental repeats and the analyses are presented in the graph. A 90° rotation negative control was employed to ensure colocalisation was not random. A Kruskal Wallis one-way ANOVA with a multiple comparisons test was performed in Prism to statistically analyse the data. Error bars represent standard deviation from the mean. Bottom right: HCT116 cells were transfected with either EV or FLAG-RALB WT for 24 h, and FLAG and DR5 expression was determined by WB. D Schematic of the role of RALB in regulating DR5 expression levels.