Effect of siRNA RUNX1 knockdown and CRISPR/Cas RAB31 knockout in megakaryocytic HEL cells on EGFR trafficking. (A) Effect of siRNA knockdown of RUNX1. Cells were exposed to EGF (0.25 μg/mL) for 8 minutes, immobilized on coverslips, and stained for EEA1 and EGFR. Compared with WT cells (upper panels), with RUNX1 knockdown (lower panels) there was marked enlargement of EEA1-positive vesicles (green) with colocalization of EGFR (red). (B) CRISPR/Cas9 RAB31 knockout (KO) cells were exposed to EGF for 8 minutes and immobilized on coverslips, and stained for EGFR (red), EEA1 (green), and CD63 (blue). Compared with WT cells (upper panels), with RAB31 KO (lower panels), there was enlargement of EEA1-positive vesicles with colocalization of EGFR, shown by yellow arrows and the insets. There was partial colocalization of EGFR with CD63. (C) Quantification of size of EEA1-positive particles in WT, RUNX1-depleted, and RAB31-depleted cells. Presented as mean ± standard error of the mean of 3 independent experiments. Scale bar = 10 µm.