Phenotypic and gene expression response of K562 and K562^Runx1KD cells to TPA treatment. (A) Quantitative RT-PCR and Western blot analysis of RUNX1 expression in untreated (U.T) and TPA-treated (TPA) cells as well as in TPA-treated cells stably expressing shRNA-miR (K562^RUNX1KD), which knocked down RUNX1 expression (KD) compared with nonsilencing shRNA-miR control (Neg). Quantitative RT-PCR data are mean ± SD of 2 independent experiments performed in triplicates. Western blot of nuclear extract using anti-RUNX1 antibodies. Emerin was used as control of protein loading (supplemental data). (B) Proliferation assay using K562-TPA Neg (red) and KD (blue) cells. A total of 1 × 10⁶ cells were grown in culture and counted every 24 hours. TPA was added 48 hours after seeding of cells. Data are mean ± SD of 2 independent experiments performed in triplicates. (C) Cell morphology changes. Representative microscopic images of May-Grunwald-Giemsa stained untreated (U.T), TPA, TPA Neg, and TPA KD K562 cells. Images were viewed with a Nikon Eclipse 800 microscope with a 100×/1.25 numeric aperture oil objective lens, captured using a Nikon DXM1200 digital camera, and processed with Nikon ACT-1 2.63 software. (D) Expression of megakaryocytic markers in K562 (Control) and K562^RUNX1KD (KD) in response to TPA. Quantitative RT-PCR analysis of ITGA2B (CD41), ITGB3 (CD61), and CD9 expression in K562 cells. Data are mean ± SD of 2 independent experiments performed in quadruplicates. The decreased expression in TPA-treated K562^RUNX1KD relative to K562 control was significant (P < .01) for all 3 markers. Primers used for quantitative RT-PCR assays are listed in supplemental Table 2. (E) TPA-induced transcriptional changes. Genes are plotted based on their expression level (log scale) in K562-TPA cells versus their basal level in untreated cells. Genes showing 2-fold (1 in log scale) increase or decrease in expression levels are indicated in green or red, respectively. Indicated are examples of up-regulated genes known to play an important role in megakaryocytic differentiation along with the reference TRPV6 gene. (F) RUNX1 knockdown impaired TPA-induced transcriptional activation. Gene expression levels in K562-TPA versus K562^Runx1KD-TPA cells, stably expressing a TPA-responsive RUNX1 shRNA-miR, are shown in supplemental Table 1. Note the change in RUNX1-dependent expression of genes indicated in panel E. (G) Change in RUNX1-dependent transcriptional regulation in TPA-treated cells. Genes were divided into groups according to differential expression after TPA treatment. Shown are boxplots representing the expression change distribution in K562^Runx1KD cells (blue) and K562^CONTROL (red) for each group of genes. In RUNX1 expression KD, transcriptional repression (left section) did not change, whereas transcriptional activation (right section) was almost completely abolished.