Figure 4.

Regulation of ER-to-Golgi transport by RUNX1 and RAB1B in HEL cells. (A) PMA-treated HEL cells cotransfected with VSVG-GFP and E2 Crimson-GalT, along with siRNAs and plasmid constructs as indicated, were seeded on coverslips, kept at 40°C for 16 hours, and then transferred to 32°C for 30 minutes and fixed. VSVG, green; GalT, red; DAPI, blue. Yellow arrowheads indicate areas of VSVG in Golgi structures, indicated by colocalization with GalT, which appears as yellow. Bar, 10 μm. VSVG and GalT were colocalized intact in the cells transfected with control siRNA (Pearson’s correlation coefficient r = 0.609 ± 0.035; mean ± SEM). In cells transfected with RUNX1 siRNA (r = 0.378 ± 0.033; P < .05) or RAB1B siRNA (r = 0.374 ± 0.026; P < .05), Golgi was disrupted and there was no accumulation of VSVG-GFP, as RAB1B facilitates VSVG transport. (B) Resumption of VSVG transport by reconstituted RAB1B in HEL cells after RUNX1 siRNA downregulation (r = 0.711 ± 0.046; P = NS compared with control). Bar, 10 μm. (C) Immunoblot analysis showing reconstitution of RAB1B protein by ectopic RAB1B expression in RUNX1-depleted HEL cells.