L-plastin regulates MK migration, podosome formation, and IMS. (A) MK migration in transwells toward the SDF-1α chemoattractant on day 13. CD41a⁺CD42a⁺ MKs quantified by flow cytometry and normalized to no SDF-1α (n = 3). (B-C) Representative F-actin staining of spread MKs on day 11 by total internal reflection fluorescence (TIRF) microscopy for (B) control MKs and (C) MKs with L-plastin knockdown. (D) Quantification of MK podosomes per MK without or with L-plastin knockdown on day 11 by TIRF (n = 3). At least 25 MKs per condition per cord were analyzed. (E) Representative image of day 14 cultured MKs were stained for actin using ActinRed 555 ready probes (shown in red) and Wiskott-Aldrich syndrome protein (WASp) using anti-WASP antibody Alexa Fluor 488 (shown in green). Scale bar, 5 μm. (F) Representative image of day 14 cultured MKs on fibrinogen were stained for L-plastin using anti-L-plastin primary antibody and Alexa Fluor 555 seconday antibody (shown in red) and WASp stained with anti-WASP primary antibody and Alexa Fluor 488 seconday antibody (shown in green). White box shows magnified image. Scale bar, 10 μm. (G-H) Representative transmission electron microscopic images showing MK IMS (red) without (G) or with (H) L-plastin knockdown. Scale bar, 5 μm. (I) Quantification of MK IMS without or with L-plastin knockdown. IMS is filled with red color using binary editor function of Nikon NIS-Element software (n = 3). At least 15 MKs per condition per cord were analyzed.