Effect on M6PR trafficking in HEL cells of RUNX1 siRNA knockdown and of RAB31-CRISPR/Cas9 knockout (KO). HEL cells were immobilized on cover slips and immunostained for M6PR (red) and EEA1 (green). Shown are images from confocal microscopy. (A) RUNX1 knockdown. (B) RAB31 KO. Under both conditions, in WT cells there is partial colocalization of M6PR with EEA1. With RUNX1 knockdown or RAB31 KO, there is colocalization of M6PR with enlarged EEA1 particles in the merged images. (C) Left panel: Immunoblotting on HEL lysates transfected with control or RUNX1 siRNA. Actin was used as loading control. Bars show densitometric analysis on immunoblot protein expression. Presented as mean ± standard error of the mean of 4 independent experiments. M6PR expression was increased in RUNX1 siRNA-depleted cells. Right panel: Immunoblotting shows M6PR expression in RAB31-KO (CRISPR/Cas) clones and actin as loading control. M6PR expression was increased in 5 of 6 clones with RAB31 KO. Scale bar = 10 µm. P values indicate comparisons vs control siRNA; *P < .001, ****P < .0001.