Impaired proplatelet formation in CIP4-deficient human megakaryocytic cell line. (A) Decreased protein expression of CIP4 and WASP after shRNA transduction of the CHRF-288-11 cells. Western blot demonstrated successful knockdown by lentiviral mediated shRNAs of CIP4 (79% reduction compared with control) or WASP (90% reduction compared with control) or TOCA1 (74% reduction compared with control). Control cells were treated with a lentiviral-mediated nontargeting shRNA sequence. (B) Morphologic changes of decreased proplatelet protrusion (arrows) and rounding up in CIP4-deficient CHRF-288-11 cells. CHRF-288-11 cells, transduced by control shRNA (upper 2 rows) were compared with CHRF-288-11 cells with CIP4 knockdown (lower 2 rows). Pictures were taken on a Nikon Biostation (objective ×20/numerical aperture 0.80; bars represents 10 um, Nikon, Tokyo, Japan) and using the Biostation IM system and dedicated software (Nikon, Tokyo, Japan). (C) Decreased proplatelet-like extensions in CHRF-288-11 cells deficient in CIP4. CHRF-288-11 cells with shRNA-CIP4 knockdown or shRNA-WASP knockdown vs control cells with nontargeting shRNA sequence were exposed to PMA at 10 ng/mL overnight. The percentage of cells with proplatelet-like extensions megakaryocytes is reported after scoring 300 cells. Compared with the control, the percentage was decreased with shRNA CIP4 (P = .04) but not for cells with shRNA WASP (P = .10; t test) nor with TOCA1 (P = .54). The data are shown as the average ± standard error of the mean from 3 independent experiments. (D) Decreased median length of proplatelet-like extensions in CIP4-deficient CHRF-288-11 cells. Protrusions were measures in at least 25 cells per condition using NeuronJ. The median length of protrusions was decreased in CHRF-288-11 cells with shRNA knockdown compared with control (P = .049; t test). Reported is average ± standard error of the mean from 3 independent experiments.