Effect of shRNA RUNX1 KD in primary human MK on albumin and fibrinogen uptake and platelet levels of Flot1, LAMP2, RAB11, and IFITM3 in patient with FPDMM. (A) Effect of shRNA RUNX1 KD in primary MK on uptake of albumin by flow cytometry. MK were differentiated in vitro from human CD34⁺ cells for 12 days. CD34⁺ cells were infected with shRX1- or shNT-lentiviruses and expressing mCherry (mCherry⁺) and sorted on day 4 of differentiation. From day 5 cells were cultured until day 11 or day 12 to mature MK and used for protein uptake studies using flow cytometry as described for HEL cells. Left panel: line graph shows MFI of albumin Alexa 488 uptake by MK overtime. Black lines indicate shNT and red shRX MK. Shown mean of 2 experiments. Right panel: representative histograms of albumin Alexa 488 uptake at the initial time point and 24 hours. (B) Effect of shRNA RUNX1 KD in MK on uptake of fibrinogen Alexa 647 by flow cytometry. Black lines indicate shNT and red shRX MK. (C) Effect of shRNA RUNX1 KD in MK on levels of RUNX1, Cav1, Flot1, LAMP2, RAB11, IFITM3, and actin (as loading control) by immunoblotting. Shown on the right is the relative protein levels done in duplicate. (D) Effect of shRNA RUNX1 KD in MK on albumin colocalization with Cav1, and Flot1 (top panels, 30 and 120 minutes) and with LAMP2 (bottom panel, 30 minutes) by immunofluorescence microscopy. Representative images are shown. shNT and shRX cells were incubated with 30 μg/mL albumin Alexa 488 for 30 and 120 minutes, fixed and immobilized on poly-L-lysine-coated coverslips. Albumin is shown in green fluorescence. In the top panels, cells were additionally stained with anti-Cav1 (red) or anti-Flot1 (blue) antibodies to assess colocalization as seen in merged images. In the bottom panel the cells were additionally stained with anti-LAMP2 (blue). (E) Platelet levels of Flot1, LAMP2, RAB11 and IFITM3 in patient with FPDMM (P, father) and 3 healthy donors (N, 1-3).