Fig. 6: RAB1A depletion impairs the ER-to-Golgi secretory pathway.
From: RAB1A is a novel vulnerability in uveal melanoma revealed by dual inhibition of MNK1/2 and mTOR
A Schematic overview of the RUSH live cell imaging assay in T128 UM cell lines (pLKO and shRAB1A#1/2). Cells are transiently transfected with a plasmid containing the hook (STR-Li: HA) and the reporter (GPI-SBP-eGFP). Newly synthesized reporters are retained in the ER (donor compartment) by the hook. Once biotin is added to the medium, reporters are released into the cytoplasm, allowing them to resume its trafficking to the Golgi. B Representative confocal images of cargo protein (GPI-SBP-eGFP, green) distribution and ER anchor protein (STR-Li:HA, red) prior to the addition of biotin (T0) in T128 cells. Single channels for Li and GPI are shown above the magnification of the boxed area displayed in the upper-right panel. C Representative confocal images of cis-Golgi (GM130, red) and cargo protein GPI-SBP-eGFP (green) at 0-, 10-, 20-, or 30-min post-addition of biotin to the culture medium. Individual and merged channels of the magnified boxed area are shown above each image. D Pearson coefficient correlation of STR-Li:HA and GPI-SBP-eGFP in c. E Pearson coefficient correlation of GM130 and GPI-SBP-eGFP at each timepoint of the RUSH assay. b, d: n = 3 for pLKO Control, shRAB1A#1 n = 3, shRAB1A#2 n = 3. c, e: n = 3. Error bars represent the mean ± S.E.M. One-way or two-way ANOVA with Dunnett post-comparison test was performed to compare RAB1A KD cells to the control. F Schematic describing the effect of decreased RAB1A expression on the secretory pathway, where it shows the improper localization of cargoes through the cytoplasm using the RUSH assay.
