Effects of modulation of HSPC111 expression on cell proliferation. (a) Immunoblot analysis of whole cell lysates from MCF-7 clones stably expressing HSPC111 (HSPC#1 and HSPC#4) or LacZ controls at various passages after transfection. Blots were analyzed for expression of tagged HSPC111 protein using V5 antibody or actin as a loading control. (b) Left panel: Growth curves of HSPC111 over-expressing clones and LacZ controls. Right panel: Stable transfectants were treated with tamoxifen (TAM), ICI 182780 (ICI), or vehicle for 48 hours, and then S phase was determined by flow cytometric analysis of propidium iodide-stained cells. (c) Upper panel: Endogenous HSPC111 mRNA and protein expression in MCF-7 cells 24 and 48 hours after transfection with HSPC111-specific small interfering (si)RNA (siHSPC2 and siHSPC4) determined by quantitative real-time PCR and immunoblot analysis with HSPC111 antibody, respectively. NS indicates mock transfection with no siRNA, RF indicates RISC-free control siRNA, and NT indicates nontargeting control siRNA. Lower panel: S phase was determined 48 hours after transfection by flow cytometric analysis of propidium iodide-stained cells.