Fig. 1

The distribution, expression and activity of HSF1. a Representative images show the cellular distributions of HSF1 in CGL2 cells at interphase (left panel) and metaphase (right panel). Logarithmically growing cells were fixed and stained with anti-HSF1 (green). The spindle microtubules were counterstained with anti-α-tubulin (red) and chromosomes with DAPI (blue). Scale bar, 10 μm. b Representative images show the cellular distributions of HSF1 phosphorylated at serine 326 (HSF1-pS326) in CGL2 cells at interphase and metaphase. Cells were immunostained with the two HSF1-pS326 antibodies shown in red (left panel, arrows) and in green (right panel, arrows) and respectively counterstained with anti-α-tubulin or anti-γ-tubulin. Arrows indicate the spots stained by HSF1-pS326 antibodies. sh-HSF1, cells depleted of HSF1 were used to test the antibody specificity and were immunostained with the corresponding HSF1-pS326 antibody (shown in green) and α-tubulin (shonw in red). c Levels of HSF1 and HSF1-pS326 at each cell cycle stage or in drug-arrested mitotic cells. CGL2 cells were enriched at each cell cycle stage by double-thymidine block and release as described in the section “Methods”, or cells were treated as indicated for 24 h. Cells were then collected by scraping the plate, or mitotic cells were collected by shaking off the plates. The collected samples were then used for immunoblot analysis. Red arrowhead indicates the shifted HSF1 band. d Transactivation activity of HSF1 at each cell cycle stage. CGL2-A1A-Luc cells were synchronized at the G1/S transition by double-thymidine block and then released from the block for the indicated times. The cells were then collected for analysis of luciferase activity assay or flow cytometry assay. 9A, the attached cells at 9 h after thymidine release. 9F, the floating cells at 9 h after thymidine release. Data shown are mean ± SD from three independent experiments. *p < 0.05 and **p < 0.01 by Student’s t-test as compared with the asynchronized cycling cells (Asyn)