Effect of siRNA KD of RUNX1, CAV1, or both on fibrinogen colocalization with Cav1, Flot1, RAB11, and LAMP2 in HEL cells by immunofluorescence microscopy. (A) Representative images showing the effect of KD of RUNX1 or CAV1 alone or in combination on fibrinogen colocalization with Cav1 and Flot1. The experimental design was same as in Figure 4. HEL-cell suspensions (control cells and those with siRNA KD of RUNX1, CAV1, or combination) were incubated with fibrinogen Alexa 647, fixed, and immobilized on poly-lysine-coated coverslips. HEL cells were additionally stained with anti-Cav1 (green) or anti-Flot1 (blue) antibodies to assess colocalization as seen in merged images, evaluated by Epifluorescence microscope (EVOS FL Autoimaging). Because HEL cells had minimal endogenous Cav1, it was enhanced to assess colocalization with albumin. At 30 and 120 minutes, in control cells fibrinogen (red) showed no colocalization with Cav1 (green) or with Flot1 (blue). With CAV1 KD alone fibrinogen uptake was unaffected compared to control cells. (B) Representative images showing the effect of KD of RUNX1, CAV1 KD, or the combination on fibrinogen (red) colocalization with RAB11 (green), a marker for recycling endosomes. At 30 minutes, there was low to moderate fibrinogen colocalization (merged images, yellow) with RAB11 in control cells, and this was increased with RUNX1 KD and with KD of RUNX1+CAV1 (left panel); the colocalization with RAB11 was more prominent at 120 minutes (right panel). (C) Representative images showing the effect of KD of RUNX1 or CAV1 KD alone or in combination on colocalization of fibrinogen with lysosomal marker LAMP2 (green). At 30 (left panel) or 120 (right panel) minutes, there was similar low colocalization of fibrinogen (red) with LAMP2 (green) in control cells or on RUNX1 KD.