Fig. 2

Heat shock modulates the NF-κB responses to IL1β stimulation. a Schematic diagram of TNFα and IL1β-dependent signal transduction pathways leading to IKK and NF-κB activation. b Schematic representation of HS and IL1β treatment: MCF7 cells were either cultured under normal conditions (37 °C) (i) or subjected to 1 h HS at 43 °C, recovered for 0, 1, 2, or 4 h (ii - v, respectively), and treated with IL1β. c The level of p65-Ser536 phosphorylation in response to HS and/or and IL1β treatment (as represented in b) assayed via Western blotting in whole cell lysates at indicated time points (min). Shown also are cytokine-unstimulated controls (0′) and β-actin loading control. d Confocal microscopy images of representative cells stably expressing p65-EGFP. Top panel: cells cultured under normal conditions (at 37 °C, no HS) and stimulated with IL1β (displayed at indicated times). Bottom panel: cells exposed to 1 h HS at 43 °C and stimulated with IL1β (displayed at indicated times post HS). Time after IL1β stimulation displayed in minutes. Scale bar 5 μm. e Nuclear NF-κB trajectories in cells stably expressing p65-EGFP for different HS and IL1β treatment protocols (as represented in b). Top: individual single cell trajectories (n = 50 per condition, in aribirary fluorescene units) depicted with green or red lines; black lines represent the population averages. Bottom: heat maps of single cell trajectories normalized across all considered conditions (represented by an arbitrary 0–3 scale). Cells monitored for up to 10 h from the beginning of IL1β stimulation. f Characteristics of single cell NF-κB trajectories from e. From the left: distribution of area under the curve (AUC), first peak amplitude, and time to first response. Individual cell data are depicted with circles (with mean ± SD per condition). Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparisons test was used to assess differences between groups (*p < 0.05, ***p < 0.001, ****p < 0.0001, ns – not significant). g Distribution of the total peak numbers between different experimental conditions