L-plastin regulates MK PPF and branching. (A) Representative immunoblot of L-plastin after CRISPR-Cas9 knockdown (k-d) in day 10 human CD34⁺-derived cultured MKs. Guide RNAs not targeting known genes are used as negative control (Ctrl). (B) Fold changes of MK L-plastin knockdown assessed by densitometry (n = 8). (C) Ctrl and CRISPR L-plastin k-d MKs on day 14 were stained with anti–L-plastin antibody (green) and imaged using a confocal microscope (60× objective). Scale bar, 10 μm. Staining for both Ctrl and k-d was performed under identical conditions and fluorescence acquisition for both images was obtained with identical microscope settings. (D) Representative brightfield images of cultured PPF MKs (white arrows) after L-plastin knockdown. Scale bar, 25 μm. (E) The percentage of MKs with PPs, scored blinded, after L-plastin knockdown (n = 9). (F) Fold changes of LCP1 expression assessed by real-time qPCR after L-plastin (L-p Lenti) or empty vector (EV Lenti) lentiviral overexpression (n = 4). (G) The percentage of MKs with PPs after L-plastin overexpression scored blinded as to the treatment group (n = 5). (E,G) At least 100 MKs per condition per cord were analyzed. (H) Brightfield image shows MK PP branches (yellow arrows) after L-plastin knockdown. Scale bar, 25 μm. (I) Quantification of MK PP branching, blinded to the treatment groups. At least 30 MKs per condition per cord were analyzed for n = 6 independent cultures.