Fig. 3

HSF1 regulates the recovery of TNFα-induced NF-κB signalling after HS. a Schematic representation of the HSR and NF-κB crosstalk. b Western blot analysis of HSF1, phosphorylated HSF1-Ser326 and HSPA1 proteins level in MCF7 cells. Cells were either cultured in normal conditions (C) or subjected to 1 h HS at 43 °C then recovered for up to 6 h. β-actin was used as a loading control. c Quantitative RT-PCR analysis of HSPA1A mRNA abundance at different time points after 1 h HS at 43 °C (normalised to the reference GAPDH gene). Shown are fold-changes (mean ± SDs of three replicate experiments) with respect to expression at 37 °C (no HS).d Effect of HSF1 knock-down on TNFα-induced response. MCF7 cells cultured in normal conditions were treated with scrambled siRNA control (scrambled) and HSF1-specific siRNA (KD HSF1) and stimulated with TNFα for indicated time periods (min). Shown are the levels of p65-Ser536, HSF1 and HSPA1 assayed via Western blotting of the whole cell lysates. Shown are cytokine-unstimulated controls (0′) and β-actin loading control. e Effect of HSF1 knock-down on IL1β-induced response in cells cultured and stimulated as in d. f Effect of HSF1 knock-down on TNFα-induced response following 4 h HS recovery. Cells treated with scrambled siRNA control (scrambled) and HSF1-specific siRNA (KD HSF1) were subjected to 1 h HS at 43 °C and recovered for 4 h, then stimulated with TNFα for indicated times (in min). Shown are the levels of p65-Ser536, HSF1, and HSPA1 assayed via Western blotting of the whole cell lysates. Shown also are cytokine-unstimulated controls (0′) and β-actin loading control. As a positive control (PC), scrambled 15′ TNFα sample from d was loaded. g Effect of HSF1 knock-down on IL1β-induced response following 4 h HS recovery in cells modified and heat-shocked as in f. As a positive control (PC), scrambled 15′ IL1β sample from e was loaded. h Effect of HSF1 knock-down on TNFα-induced NF-κB response in MCF7 cells stably expressing p65-EGFP. Cells treated with scrambled siRNA control (scrambled) and HSF1-specific siRNA (KD HSF1) were either cultured in normal conditions (37 °C, no HS) or subjected to 1 h HS at 43 °C and recovered for 4 h (1 h HS + 4 h). Individual single cell nuclear p65-EGFP trajectories (n = 202, 127, 146, 98 respectively, in aribirary fluorescene units) are depicted with blue and violet lines; black lines represented the population averages. Cells monitored for up to 10 h from the beginning of cytokine stimulation. i Effect of HSF1 knock-down on IL1β-induced NF-κB response in MCF7 cells stably expressing p65-EGFP. Cells were cultured and stimulated as in h (n = 153, 140, 113, 42 respectively). For heat maps and number of responding cells see Fig. S2