Figure 2.
Figure 2. HSF2 represses the HSR in mitotic cells. (A, top) Representative immunoblot (n = 3) of Hsp70 and Hsp25 proteins upon heat shock in HSF2 WT and HSF2 KO MEFs at different phases of the cell cycle. Unsynchronized (U), mitotic (M), or G1-phase (G1) cells were left untreated (C) or heat-treated (HS+R, 1 h at 43°C, 3 h at 37°C). Cells were synchronized into mitosis by a thymidine-nocodazole block, and mitotic cells were harvested by mitotic shake-off. G1 phase cells were obtained by releasing mitotic cells into normal growth media for 3 h. Immunoblotting was performed with the indicated antibodies. <, HSF2; *, an unspecific band. Hsc70 serves as a loading control. (A, middle) Quantifications of Hsp70 protein induction in mitotic cells (n = 3). Hsp70 was normalized to Hsc70 and then related to the respective untreated mitotic samples, arbitrarily set to a value of 1. (A, bottom) Representative flow cytometry analyses of propidium iodide–stained cells. The data shown are from a single representative experiment out of three repeats. For the experiment shown, n = 10,000. (B, top) A representative immunoblot of K562 cells transfected with the indicated shRNAs and then either left unsynchronized (U) or synchronized into mitosis (M) by a thymidine-nocodazole block. Cells were treated with a 30-min heat shock at 42°C (HS) or a 30-min heat shock followed by a 1-h recovery at 37°C (HS+R). Samples were analyzed with the indicated antibodies and tubulin was used as a loading control. (B, middle) Quantifications of Hsp70 protein induction in mitotic cells (n = 5). Hsp70 was normalized to tubulin and analyzed as in A. (B, bottom left) Flow cytometry analyses of propidium iodide–stained cells. The data shown are from a single representative experiment out of five repeats. For the experiment shown, n = 10,000. (B, bottom right) The mitotic index was determined based on chromosome morphology of cells DNA stained with Hoechst. 80 cells per treatment from three independent experiments were counted. (C) qRT-PCR analysis of hsp70 mRNA induced by heat shock (30 min at 42°C) in mitotic K562 cells. Cells were transfected and synchronized as in B. Relative quantities of hsp70 mRNA were first normalized to gapdh and then calculated from the respective untreated samples, arbitrarily set to a value of 1. Values represent mean + SEM (error bars; n = 5). n.s., not specific.

HSF2 represses the HSR in mitotic cells. (A, top) Representative immunoblot (n = 3) of Hsp70 and Hsp25 proteins upon heat shock in HSF2 WT and HSF2 KO MEFs at different phases of the cell cycle. Unsynchronized (U), mitotic (M), or G1-phase (G1) cells were left untreated (C) or heat-treated (HS+R, 1 h at 43°C, 3 h at 37°C). Cells were synchronized into mitosis by a thymidine-nocodazole block, and mitotic cells were harvested by mitotic shake-off. G1 phase cells were obtained by releasing mitotic cells into normal growth media for 3 h. Immunoblotting was performed with the indicated antibodies. <, HSF2; *, an unspecific band. Hsc70 serves as a loading control. (A, middle) Quantifications of Hsp70 protein induction in mitotic cells (n = 3). Hsp70 was normalized to Hsc70 and then related to the respective untreated mitotic samples, arbitrarily set to a value of 1. (A, bottom) Representative flow cytometry analyses of propidium iodide–stained cells. The data shown are from a single representative experiment out of three repeats. For the experiment shown, n = 10,000. (B, top) A representative immunoblot of K562 cells transfected with the indicated shRNAs and then either left unsynchronized (U) or synchronized into mitosis (M) by a thymidine-nocodazole block. Cells were treated with a 30-min heat shock at 42°C (HS) or a 30-min heat shock followed by a 1-h recovery at 37°C (HS+R). Samples were analyzed with the indicated antibodies and tubulin was used as a loading control. (B, middle) Quantifications of Hsp70 protein induction in mitotic cells (n = 5). Hsp70 was normalized to tubulin and analyzed as in A. (B, bottom left) Flow cytometry analyses of propidium iodide–stained cells. The data shown are from a single representative experiment out of five repeats. For the experiment shown, n = 10,000. (B, bottom right) The mitotic index was determined based on chromosome morphology of cells DNA stained with Hoechst. 80 cells per treatment from three independent experiments were counted. (C) qRT-PCR analysis of hsp70 mRNA induced by heat shock (30 min at 42°C) in mitotic K562 cells. Cells were transfected and synchronized as in B. Relative quantities of hsp70 mRNA were first normalized to gapdh and then calculated from the respective untreated samples, arbitrarily set to a value of 1. Values represent mean + SEM (error bars; n = 5). n.s., not specific.

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