Effect of sustained suppression of HDAC2 in MKN-1 cells on in vitro tumorigenicity. Effect of sustained suppression of HDAC2 in MKN-1 cells on in vitro tumorigenicity. A, Western blot analysis of HDAC2, p16INK4a and cyclin D1 in total lysates of MKN-1 cells with stable expression of short hairpin RNA (HDAC2 KD1 and HDAC2 KD2) or mock (Empty vector, pSilencer 2.1). Note the considerable HDAC2 protein depletion in the cell lines. HDAC2 gene silencing restored p16INK4a expression but repressed cylin D1. α-tubulin expression served as loading control. The data are at least n = 3 independent experiments. B, cell proliferation was determined by the MTT assay. Data are mean ± SD. *, P < 0.01. C, cell migration assay. The assay was conducted in the Boyden chamber; here, NIH/3T3 cells were used as a positive control (= mock). The graph depicts the average number of cells migrating of n = 3 independent experiment; data are means ± SD. *, P < 0.01. D, the invasive abilities of the indicated cell lines were examined by the Transwell invasion assays. *, P < 0.01 versus mock cells. E, anchorage-independent colony growth was visualized by staining with crystal violet; colonies were counted and analyzed. Data are means ± SD. *, P < 0.01. F, left, specific senescence-associated marker, β-galactosidase (SA-β-gal), was determined by X-gal staining. Right, the bar graph shows the average number of senescent cells as a % of total cell that were positively stained by SA-β-gal. Results are means ± SD. *, P < 0.01. Jeong Kyu Kim et al. Mol Cancer Res 2013;11:62-73 ©2013 by American Association for Cancer Research