HSF2 protein and mRNA levels decrease during mitosis. (A, left) Representative immunoblot analysis of HSF2 protein in unsynchronized K562 cells (U), cells synchronized into mitosis by a thymidine-nocodazole block (M), G1 by releasing mitotic cells for 5 h (G1), S phase by a double-thymidine block (S), or G2 by releasing S phase cells for 5 h (G2). Immunoblotting was performed with the indicated antibodies. Tubulin serves as a loading control. The asterisk denotes phosphorylated Cdc27, which serves as a control indicating that cells are in mitosis. (A, right) Quantification of immunoblots (n = 3) showing HSF2 protein levels related to tubulin. (B) qRT-PCR analysis of hsf2 mRNA in unsynchronized (U) and mitotic (M) K562 cells. Relative quantities of hsf2 mRNA were first normalized to gapdh, and then unsynchronized samples were arbitrarily set to a value of 1 (n = 5). (C, left) Immunoblot analysis of HSF2 protein in unsynchronized (U) and mitotic (M) K562 cells. Cells were left untreated (C) or subjected to heat shock (HS; 30 min at 42°C, 1 h at 37°C). (C, right) Quantifications of blots where HSF2 was related to Hsc70 (n = 4). (D) Representative immunoblot analysis of HSF2 protein in K562 cells (n = 3). Cells were either left unsynchronized (U); treated with thymidine (24 h), followed by, after a 4-h release, the addition of nocodazole for 3 h (Thy-Noc); only treated with nocodazole for 3 h (U+Noc); or released from thymidine for 6.5 h (Thy+6.5 h). (D, right) Flow cytometry analysis of propidium iodide–stained cells. The data shown are from a single representative experiment out of three repeats. For the experiment shown, n = 4,000. All values represent mean + SEM (error bars).