Figure 1.

Hsp27 accelerates cell growth by promoting ERK nuclear translocation in LNCaP cells. (a) Hsp27 accelerates cell growth. Cell growth rates of LNCaP_Hsp27 and LNCaP_mock were compared by direct counting of viable cells. Levels of total and phospho-Hsp27 were analyzed by immunoblotting. (b) LNCaP_Hsp27 cells exhibit enhanced proliferation. Cell proliferation of LNCaP_mock and LNCaP_Hsp27 cells was assessed by [³H]-thymidine incorporation into DNA. The symbol ‘^*' denotes statistical significance (P<0.05). (c) LNCaP_Hsp27 cells display an expression profile of cell cycle markers that is consistent with increased cell proliferation. Cell lysates from LNCaP_mock and LNCaP_Hsp27 cells were analyzed for cyclin D1, p27kip1 and CDK2 by immunoblotting. Vinculin immunoblotting was performed as a loading control. (d) Hsp27 silencing inhibits LNCaP cell proliferation. Cell number was quantified using CyQuant proliferation assay (top), and percentage of cells undergoing DNA replication was determined by flow cytometric analyses of BrdU incorporation (bottom). (e) Overexpression of Hsp27 promotes ERK phosphorylation in LNCaP cells. Levels of phospho-ERK and total ERK protein were analyzed by immunoblotting. Vinculin immunoblotting was performed as a loading control. Relative levels of phospho-ERK expressions normalized to vinculin levels were analyzed in triplicate. The symbol ‘^*' denotes statistical significance (P<0.05). (f) Hsp27 stimulates Elk-1 activity in LNCaP cells. LNCaP_mock and LNCaP_Hsp27 cells were transfected with the Elk-1 transcription reporter vector (pElk-1-GAL4) at various doses (0–0.8 μg). At 48 h after transfection, the cell lysates were assayed for luciferase reporter activity. (g) Hsp27 overexpression enhances nuclear translocation of ERK. The nucleocytoplasmic distribution of ERK was assessed in LNCaP_mock and LNCaP_Hsp27 cells by immunofluorescence microscopy. Cells were immunostained with anti-Hsp27 (green) and anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used to define the cell nuclei. The mean fluorescence ratios FN/C are shown for ERK. Values shown represent mean±S.D. The symbol ‘^*' denotes statistical significance (P<0.05). The results (a, b, d and e) are the means of three independent experiments