PIP4Kα shRNA induces absence of large MKs. (A) Mouse fetal liver cultures were treated with retroviruses that drive expression of PIP4Kα-specific shRNA and selected in puromycin. Cell fractions enriched for MK progenitors were isolated on day 5 and cultured further under drug selection. MKs from the ensuing (second) wave were used to prepare protein lysates and immunoblotted with PIP4Kα antiserum to reveal substantial down-regulation of the target protein compared with controls (empty vector). Protein loading was verified by Coomassie blue staining (right; only part of the gel is shown). (B) Selectivity of PIP4Kα shRNA. The PIP4Kα antibody recognizes PIP4Kα better than PIP4Kβ. Mouse embryonic fibroblasts (MEFs) from wild-type or PIP4Kβ-nullizygous mice were treated with the same PIP4Kα shRNA. Both kinase isoforms are present in wild-type MEFs, whereas PIP4Kβ–/– MEFs only express PIP4Kα. The shRNA specifically depletes PIP4Kα without affecting the PIP4Kβ isoform. (C) Progenitor fractions were monitored for MK differentiation. PIP4Kα shRNA-treated cells failed to develop into the largest MKs, as judged by both phase-contrast light microscopy (top) and 2-parameter flow cytometry (bottom) for forward light scatter (x-axis; FSC) and surface CD41 (y-axis). (D-E) Similar studies were conducted with EYFPki MKs. Representative micrographs show typical membrane staining of MKs treated with control (empty) virus (D), whereas in PIP4Kα-depleted MKs (E), the cytoplasmic fluorescent signal is uniform and lacks definition corresponding to internal membranes. Scale bars, 15 μm.
