Radioresistance induced by heat shock is reduced in cells that lack a functional HSF1. A1-5 cells, ALTR12 cells and ALTR12 cells that constitutively express an activated HSF1 (ALTR12/ACT HSF1) were plated as described in the Materials and Methods, and the cells were exposed to increasing doses of ionizing radiation. In a parallel experiment, A1-5 and ALTR12 cells were subjected to a heat shock (42°C for 30 min) and then irradiated. All plates were incubated at 37°C for ~10 days. Surviving colonies were stained with crystal violet and quantified on a colony counter. The graph shows the average surviving fractions ± SE from three independent experiments.

Suppression of HSF1 inhibits radiation-induced 53BP1 focus formation in A1-5 cells. Panel A: A1-5 cells grown on 30-mm dishes were transfected with the indicated siRNAs as described in the Materials and Methods. Total cell extracts were prepared 48 h after the last transfection. Then 50 μg of total protein mixed with loading buffer was applied to a 10% SDS-polyacrylamide gel for analysis of HSF1 protein levels by Western blotting using an anti-HSF1 antibody. The filter was probed for β-actin to control for loading variations. Panel B: In a parallel experiment, A1-5 cells were grown on cover slips and transfected with the indicated siRNAs. Then the cells were either exposed to 10 Gy ionizing radiation or left untreated. After incubation at 37°C for an additional 2 h, cells were fixed and stained for 53BP1 by indirect immunofluorescence using an anti-53BP1 antibody. A typical result is shown. The scale bars represent 5 μm. Panel C: The fractions of cells in panel B that exhibited 53BP1 focus formation were quantified and graphed. The values are averages ± SE from three independent experiments.

The quantity of 53BP1 protein is decreased in cells that lack a functional HSF1. Panel A: A1-5, ALTR12 and ALTR12/ActHSF1 cells were grown to 60% confluence and then either left untreated or exposed to 5 Gy of ionizing radiation (IR), and incubation continued at 37°C for 2 h. The cells were harvested and fractionated into nuclear and cytoplasmic fractions. Then 50 μg of total protein mixed with loading buffer was applied to a 10% SDS-polyacrylamide gel for analysis of 53BP1 protein by Western blotting using an anti-53BP1 antibody. The blots were also probed for histone H1 (a nuclear protein) or α-tubulin (a cytoplasmic protein) to assess the purity of the nuclear/cytoplasmic fractions. Panel B: A1-5, ALTR12 and ALTR12/ActHSF1 cells were grown as described in panel A and were either left untreated or exposed to 5 Gy of ionizing radiation and incubation was continued at 37°C for 2 h. Total RNA was extracted and 53BP1 mRNA measured using quantitative RT-PCR. The PCR products of 53BP1 and GAPDH separated on a 1% agarose electrophoresis and stained with ethidium bromide are shown. Panel C: The PCR products in panel B were analyzed using the 2^−ΔΔCT quantitative PCR method. No apparent differences were seen in the 53BP1 gene expression among cell lines. Error bars represent SD for triplicate reactions. Fold change = relative change.

Lack of HSF1 results in a defective G₂ checkpoint and DNA repair in response to ionizing radiation. Panel A: A1-5, ALTR12 and ALTR12/ACT HSF1 cells were exposed to increasing doses of ionizing radiation as indicated. The cells were then incubated with nocodazole at 37°C for ~10 h and the fractions of mitotic cells were determined. The graph shows the average values normalized to the mitotic index for unirradiated cells ± SE determined from at least 500 individual cells. Panel B: A1-5, ALTR12 and ALTR12/ACT HSF1 cells were exposed to 10 Gy of ionizing radiation, incubated at 37°C, and then prepared for comet assay at various times after irradiation. The graph shows the average Olive tail moment, which was normalized to time zero. The values are averages ± SE from 100 individual cells. Panel C: A1-5, ALTR12 and ALTR12/ACT HSF1 cells were grown on cover slips at 37°C to 80% confluence and then exposed to 10 Gy or left untreated. Incubation was continued at 37°C for 2 h before fixing and staining for 53BP1 localization. The scale bars represent 5 μm. Panel D: The fraction of cells in panel C that exhibited 53BP1 foci was quantified and graphed. The values are averages ± SE from three independent experiments.

Suppression of HSF1 in Hct116 cells inhibits DNA repair and G₂ checkpoint activation in response to ionizing radiation. Panel A: HCT116 cells grown on 30-mm dishes were transfected with the indicated siRNAs as described in the Materials and Methods. Total cell extracts were prepared 48 h after the last transfection. Then 50 μg of total protein mixed with loading buffer was applied to a 10% SDS-polyacrylamide gel for analysis of HSF1 protein levels by Western blotting using an anti-HSF1 antibody. The filter was probed for β-actin to control for variations in loading. Panel B: Hct116 cells were grown on cover slips and transfected with siRNAs as indicated and either not treated further or exposed to 10 Gy ionizing radiation. After incubation at 37°C for an additional 2 h, the cells were fixed and stained for 53BP1 by indirect immunofluorescence using an anti-53BP1 antibody. The scale bars represent 5 μm. Panel C: The fractions of cells in panel B exhibiting 53BP1 foci were quantified. The graphs show the averages ± SE from three independent experiments. Panel D: HCT116 cells were transfected with siRNAs as indicated and then exposed to increasing doses of ionizing radiation. The cells were blocked with nocodazole, and the fraction of mitotic cells was determined. The graph shows the average fraction of mitotic cells from at least 100 individual cells normalized to the fraction of mitotic cells in unirradiated samples ± SE. Panel E: HCT116 cells were transfected with the siRNAs as indicated and then exposed to 10 Gy ionizing radiation. The cells were harvested at regular intervals after irradiation and prepared for the comet assay. The graph shows the average relative Olive tail moment ± SE for at least 100 individual cells.