(A) Total RNA was isolated from HEL-Ctrl(–) (white bars) and HEL-Tsc(–) cells (gray bars). Gene expression was analyzed by RQ-PCR (TaqMan) with tescalcin-, GPIIb-, Ets-1–, Ets-2–, Fli-1–, PU.1-, GATA-1–, and MafB-specific primers as described in Methods. Data are expressed relative to wild-type HEL cells (n = 3; mean ± SD). (B) Control HEL-Ctrl(–) and tescalcin knockdown HEL-Tsc(–) cells were cultured in absence or presence of PMA for 72 hours. Expression of Ets-1 and Fli-1 transcription factors was assessed by Western blot with specific antibodies. (C) Total RNA was isolated from K562-Ctrl(+) and tescalcin overexpressing K562-Tsc(+) cells cultured in absence or presence of PMA for 18 hours. RQ-PCR using GPIIb-, Ets-1–, Ets-2–, and GATA-1–specific primers was performed as described in A. Data are expressed relative to wild-type unstimulated K562 (n = 3; mean ± SD). (D) Cytospin preparations of K562 cells on glass slides were stained with antibody to tescalcin and then with FITC-labeled donkey anti-rabbit antibody. Slides were mounted with DAPI containing mounting media (Prolong; Molecular Probes) to identify cell nuclei. All images were acquired at ×1,000 magnification. Scale bar: 10 μm. (E) Outline of tescalcin-mediated pathway. TPO binds to its receptor, c-Mpl, on the cell surface and activates the ERK signaling cascade. Treatment of cells with PMA leads to a similar activation of ERK. The sustained ERK activity causes upregulation of tescalcin, which in turn promotes expression of the Ets family genes, orchestrating terminal differentiation along megakaryocytic lineage.