Impaired proplatelet formation in CIP4-null MKs. (A) CIP4-null MKs form less proplatelets. CIP4-null, WASP-null, and WT megakaryocytes were grown in culture in suspension with TPO. At day 4, the percentage of proplatelet-forming MKs was reported by scoring 300 MKs. The data shown are the average ± standard error of the mean from 3 independent experiments, except for WASP KO, which was performed twice. (B) CIP4 KO cells are mechanistically able to extend tubulin loops in their proplatelets tips. The cells were stained with immunofluorescence on day 4 or 5 of culture with TPO for β-tubulin (Alexa-488 conjugated secondary antibody) to look for ability to form loops, for von Willebrand Factor (Alexa-594 conjugated secondary antibody), to document their megakaryocytic nature, and with 4,6 diamidino-2-phenylindole to show the nucleus. Top: pictures show WT megakaryocytes. Bottom: pictures show CIP4-null megakaryocytes. Last column: pictures show proplatelets detached from the bodies of megakaryocytes. The pictures were taken with a Leica DM 4000B microscope, visualized with a Leica DFC320 camera, and analyzed by using LAS software (Leica, Wetzlar, Germany). (Objective 100×/numerical aperture 1.3; bars represent 10 um).