Fig 2: Reduction of EGFP-STAT5A(E18?) clearance by inhibition of SQSTM1 phosphorylation. (A) For formation of EGFP-STAT5A(E18?) aggregates, HeLa TREx-EGFP-STAT5A(E18?) cells were cultured in medium containing tetracycline and 2.5 µM MG132 plus mock, or kinase inhibitors (rapamycin, CKI-7, and TBCA), or KRIBB11. The medium was replaced with normal medium containing mock, or kinase inhibitors, or KRIBB11 and cells were fixed after 0, 12, or 24 h, followed by DAPI staining. The images were obtained by fluorescence microscopy. Scale bar: 100 µm. (B) EGFP-STAT5A(E18?) intensities were measured by an IN Cell Analyzer 2200. Values of EGFP intensity in individual cells were plotted on graphs (right), which show means ± SD. Statistical analyses were performed using one-way ANOVA, followed by the Tukey post-hoc test. *P< 0.01. Cell count (%) was plotted against EGFP-STAT5A(E18?) intensity (right). Upper, middle, and lower graphs show 0 h, 12 h, and 24 h, respectively. (C) Expression levels of ATG5 and ATG7 in HeLa_WT (open boxes), KRIBB11-treated HeLa (gray boxes), and HeLa HSF1 KO (closed boxes) cells were measured by real-time PCR. Relative expression values of target genes were normalized against the housekeeping gene GPD/G3PDH (glycerol-3-phosphate dehydrogenase) and are reported as means ± SD (n = 4). Statistical analyses were performed using one-way ANOVA, followed by the Tukey post-hoc test. (D) Autophagosome formation in HeLa_WT, kinase inhibitors (rapamycin, CKI-7, and TBCA) treated HeLa, KRIBB11-treated HeLa, and HeLa HSF1 KO cells was immunocytochemically examined using an anti-LC3 antibody. Cells were cultured in normal conditions (left panels), starvation conditions (middle panels), and with MG132 (right panels) for 5 h. Cell nuclei were counterstained blue with DAPI. Scale bar: 10 µm. a.u., arbitrary units.

Fig 3: ß-catenin stimulates HSF1 protein translation. a The effect of Pyrvinium on the protein expression of HSF1 was explored by western blotting. b The effect of ß-catenin knockdown on HSF1 protein level was analyzed by western blotting. c The protein level of HSF1 before and after ß-catenin overexpression was analyzed by western blotting. d The effect of LiCl on HSF1 protein level in RKO and MEF was analyzed by western blotting. e The expression of ß-catenin and HSF1 in colorectal tissue was analyzed by immunohistochemistry staining. f The correlation between ß-catenin expression and HSF1 expression in colorectal tissue was analyzed by chi-square test (*p < 0.01). g The effect of ß-catenin depletion on HSF1 with puromycin labeling was determined by western blotting. h Amount of HSF1 mRNA in various polysome fractions was analyzed by RT-PCR(*p < 0.05)

Fig 4: HSF1 regulates the expression of LRP1B and lipid metabolism in HCC cells. (A) Schematic representation of the sites of HSF1-binding in the LRP1B promoter region. The in-frame splicing indicates the sequences of LRP1B with an amino acid changed or not, where HSF1 bound at the transcription site. TSS: transcription site. ch2.2: chromosome 2.2. WT: wildtype; MUT1: mutation1; MUT2: mutation2. (B) Dual luciferase reporter assay was used to examine the effect of HSF1 on LRP1B promoter activity, **p<0.01 vs control, #p<0.05 vs WT. Control: HSF1+pGL3-basic+pRL-TK; WT: HSF1+LRP1B pro (wildtype) +pRL-TK; MT1: HSF1+LRP1B pro (MUT1) +pRL-TK; MT2: HSF1+LRP1B pro (MUT2) +pRL-TK. (C) Western blotting confirmed the efficiency of HSF1 knockdown in Huh7 cells. ß-actin was regarded as an internal control. (D) HSF1 overexpression in Huh7 cells was measured by Western blotting. ß-actin was regarded as an internal control. (E) Representative images of DiD staining in LRP1B-knockdown Huh7 cells with HSF1 overexpression. The red indicated the positive lipophilic cells (×100). (F) The percent of positive lipophilic cells collected from the same experiments as shown. (G) Western blotting was used for detecting the LRP1B transcriptional activation in Hep3B with HSF1 knockdown. (H) Representative images of DiD staining in Huh3B cells with transcriptional activation (×100). (I) The percent of positive cells collected from the same experiments as shown. *P<0.05. Data represented the mean±SD. Statistical significance was analyzed by two-tailed unpaired Student’s t-test. Triplicate experiments independently with similar results.

Fig 5: Effects of HSF1 agonist 17-AAG on the expression of C3 and S100A10 in the astrocytes. a Toxic assay of 0-10 µmol/L 17-AAG for the astrocytes. b–e Western blot analysis of C3 (b, c), S100A10 (b, d) and HSP70 (b, e) in astrocytes following cell incubation at 0–10 µmol/L 17-AAG for 24 h. Quantities were normalized to endogenous ß-actin. Experiments were performed in triplicates. Error bars represent the standard deviation, *P < 0.05, one-way ANOVA with Dunnett’s post hoc test. For C3 quantitative analysis, F (4, 10) = 4.763, P = 0.0207. 0.1 vs con, P = 0.0019; 0.5 vs con, P = 0.0526; 2.5 vs con, P = 0.0432; 10 vs con, P = 0.0066. For HSP70 quantitative analysis, F (4, 10) = 10.89, P = 0.0011. 0.1 vs con, P = 0.0051; 0.5 vs con, P = 0.0032; 2.5 vs con, P = 0.0017; 10 vs con, P = 0.0004