Effect of siRNA KD of RUNX1, CAV1, and FLOT1 on uptake and retention of fibrinogen in HEL cells. (A) Effect of siRNA KD of RUNX1, CAV1, or combination of both on uptake and retention of fibrinogen. PMA-treated HEL cells were transfected with siRNAs (100 nM): control (black dots), RUNX1 (red), CAV1 (orange), or combination of RUNX1 + CAV1 (green) for 48 hours. Cells suspensions were incubated with 10 μg/mL fibrinogen Alexa 647 for indicated times at 37°C, fixed and uptake was evaluated by flow cytometry. Shown mean ± SEM of 3 experiments. P values represent comparisons with control at 24 hours. Representative histograms are shown on the right. (B) Effect of siRNA KD of RUNX1, FLOT1, or combination of both on uptake and retention of fibrinogen by flow cytometry. RUNX1 and FLOT1 KDs were performed as described above. HEL cells were incubated with fibrinogen Alexa 647 (10 μg/mL) for indicated times. Data shown are mean of 2 experiments. Control cells (black), RUNX1 KD (red), FLOT1 KD alone (orange), combined RUNX1, and FLOT1 KD (green). Representative histograms are shown on the right. (C) HEL-cell fibrinogen levels using immunoblotting after siRNA RUNX1 KD. Cells treated with control and RUNX1 siRNAs were washed and incubated in culture media containing fetal bovine serum and 50 μg/mL fibrinogen for 24 hours. Fibrinogen levels were assessed in washed cell lysates. A representative immunoblot showing fibrinogen and RUNX1 is shown with quantification on the right (n = 4 experiments). (D) Fibrinogen levels over 24 hours in HEL cells treated with control or RUNX1 siRNAs and monitored in buffer without fibrinogen. HEL cells treated with RUNX1 or control siRNAs were washed and resuspended in media with added 10% FBS and 50 μg/mL fibrinogen for 24 hours at 37°C. Cells were washed, resuspended in media without fibrinogen or FBS. Fibrinogen levels were assessed in lysates by immunoblotting at time points shown. Shown is a representative immunoblot and quantification from 4 separate experiments.