Pim-1 knockdown cells are resistant to the effects of 2′-HCA. A, Knockdown of Pim-1 causes resistance to the inhibitory effects of 2′-HCA against anchorage-independent growth of A431 or HEL cells or EGF-induced neoplastic growth of HaCaT cells. A soft agar assay was performed with Mock- or shPim1-transfected (a) HEL, (b) A431, or (c) EGF-induced HaCaT cells treated with 2′-HCA (0, 2.5, 5, or 10 μM). The number of colonies was counted under a microscope with the aid of the Image-Pro Plus software program (vs. 6.2). The untreated control was calculated as 10% of Mock- or shPim-1-transfected HEL and A431 cells and the EGF-treated group was calculated as 100% of Mock- or shPim-1-transfected HaCaT cells. B, Growth was evaluated by MTS assay using Mock- or shPim1-transfected (a) HEL and (b) A431 cells treated with 2′-HCA (0, 2.5, 5, or 10 μM) for 48 h. C, Apoptosis was analyzed by annexin V/PI staining of Mock- or shPim1-transfected (a) HEL and (b) A431 cells treated with (0, 2.5, 5, or 10 μM) 2′-HCA in for 24 h. The asterisks (*, **) indicate a significant difference (p < 0.05, p < 0.01) between Mock- or shPim-1-transfected HEL and A431 cells.