Fig. 5: Impact of preventing or mimicking acetylation of lysine residues K128, K135, and K197 on HSF2 protein stability.

a Representative immunoblots. The inhibition of CBP/EP300 decreases HSF2 and is counteracted by proteasome inhibition in N2A cells treated with the CBP/EP300 inhibitor C646 (40 µM, 4 h) and/or with MG132 (20 µM, 6 h) (n = 4). Quantification of HSF2 signal intensity, normalized by HSC70 and relative to vehicle-treated samples (−). Error bars, mean ± standard error of the mean (SEM), *p = 0.0022. b Scheme of the principle of SNAP-TAG pulse-chase experiments. c Representative electrophoresis images of protein extracts from HSF2KO or WT U2OS cells expressing SNAP-tagged WT, 3KQ or 3KR HSF2, SNAP-labeled, and showing the decay of 3KQ HSF2 mutant protein levels (0–5 h). SNAP-H3.3, loading control. d Quantification of the fluorescent signal normalized to H3.3 and relative to the signal at t₀ (n = 7 with replicates). Error bars, mean ± SEM, p = 0.0112 (1h30,KQ vs.WT), p = 0.0029 (1h30,KR vs.KQ), p = 0.0045 (3 h,KQ vs.WT), p = 0.0039 (3 h, KQ vs. KR), p = 0.0076 (5 h, KQ vs. WT); p = 0.0015 (3 h, KQ vs.KR) *p < 0.05; **p < 0.01. e Representative electrophoresis images of protein extracts from Hsf2KO U2OS cells expressing SNAP-tagged HSF2 WT or 3KR, pretreated by MG132 (vehicle (0), 10, 20 µM, 5 h), SNAP-labeled, and analyzed after 5 h, showing the decrease in SNAP-HSF2 WT and 3KR protein levels depending on proteasome activity (n = 3). Error bars, mean ± SEM. p = 0.0286 (KR vs. KR_MG), p = 0.0159 (WT vs. WT_MG) *p < 0.05, quantification as in d. f Representative immunoblots of immunoprecipitated HSF2-Myc (IP Myc) from HEK 293 cells transfected with HSF2-Myc WT, 3KR, or 3KQ, and treated (+) or not (−) with MG132 (20 µM, 6 h), showing preferential poly-ubiquitination of the HSF2 3KR mutant protein, compared to HSF2 WT or 3KQ (n = 3). g Representative electrophoresis image as in c, but Hsf2KO U2OS cells were treated with HS (42 °C) or not, (CTR), and analyzed prior (0, grey) or after 2.5 h (light grey) of HS (red), showing increased HSF2 3KQ stability, upon HS, compared to WT or 3KR (n = 7). Error bars, mean ± SEM *** p = 0.0011 (3KQ, HS 2h30 vs. CTR 2h30)), ****p < 0.0001 (WT, HS 2h30 vs. CTR 2h30); p = 0.0001 (3KR, HS2h30 vs. CTR 2h30). Quantification as in d. Significance was calculated by two-sided Mann–Whitney test in panels a, d, e, g. Source data are provided as a Source Data file.