Fig. 1: RASMT CRC cells are dependent on RALB for survival.

A Left: Crystal violet cell viability assay in KRASMT and KRASWT cells following transfection with 10 nM scrambled control (SC), RALA (siRALA) or RALB (siRALB) siRNA for 72 h. Cell viability values are presented relative to SC and the dashed line represents a 25% decrease in viability. Right: Clonogenic survival assay in HCT116 cells following transfection with 1 μg empty vector (EV), RALB wild-type (WT) or RALBG23V constructs and incubation for 14 days. Western blot (WB) analysis of FLAG-tag carried out in HCT116 cells transfected with EV, RALB-WT and RALBG23V constructs. β-actin was used as a loading control. B Top: WB analysis of PARP, RALA, RALB, pERK1/2^T202/Y204 and ERK1/2 expression in HCT116 and LIM1215 cells, following transfection with SC, siRALA or siRALB and co-treatment with 1 μM AZD6244 for the indicated time. Bottom: Apoptosis was assessed by propidium iodide (PI) flow cytometry and Caspase-3/7 activity levels in HCT116 and LIM1215 cells, following transfection with SC, siRALA or siRALB and co-treatment with 1 μM AZD6244 for 24 h. C SW620 and GP5d cells were transfected with SC, siRALA or siRALB and co-treated with 1 μM AZD6244. PARP, RALA, RALB, pERK1/2^T202/Y204 and ERK1/2 expression was determined by WB and β-actin was used to assess equal loading. Apoptosis was assessed by PI flow cytometry and by measuring Caspase-3/7 activity. D Top: WB analysis showing RALB expression in matched CRC (T) and normal (N) tissues. Equal loading was assessed by analysing β-actin expression. Bottom: Boxplots representing RALB protein abundance across CRIS subgroups in the COREAD dataset. E Boxplots representing the log2 gene expression values for RALB (probes ADXECAD.28315_at and 202101_s_at) across CRIS subgroups in GSE103479 and GSE14333 clinical datasets. Numbers underneath the boxplots (n) indicate the sample number per group.