Fig 1: HSF1 is activated during fat accumulation in the NAFLD model. To evaluate whether PA and OA induce nuclear HSF1 binding to HSEs, we conducted gel shift assays with oligonucleotides harboring a consensus HSE in HepG2 and Hep3B cells (a). For supershift experiments, nuclear extracts of Hep3B cells were treated with two antibodies directed against different sites of HSF1 (b). Representative results of four independent experiments are shown.
Fig 2: Regulation of HSF1 Target Genes by 14a-Stabilized DHFR.dn-cHSF1 in HEK293T-Rex Cells(A and B) The expression levels of (A) HSPA1A and (B) DNAJB1 under different treatment conditions, normalized to vehicle (DMSO)-treated HEK293T-Rex control cells stably expressing DHFR.YFP. Data presented are the mean ± SD of technical triplicates. (C) Western blot probing endogenous HSF1 and DHFR.dn-cHSF1 in cells treated with DMSO, 10 µM TMP, or 14a for 24 h. ß-actin was used as the internal control. Representative image of three independent experiments.
Fig 3: HSF1 staining of hepatocytes before and after bariatric surgery. Representative images of immunohistochemical staining of HSF1 in liver tissue obtained during bariatric surgery ((a), left panel) and 6 to 12 months after bariatric surgery ((a), right panel) (scale bar 100 μm) are shown. The expression score (ES = P × S) ± mean of HSF1 nuclear staining of 9 liver tissues is depicted (b).
Fig 4: HSF1 binds to a putative HSF1 binding site in the CPT1a promoter. Sequence analysis identified a putative binding site for HSF1 (labeled in red) in the CPT1a promoter (a). Nuclear extracts from F+ cells were analyzed by gel shift assays with oligonucleotides that spanned the putative HSF1 binding site (lane 1,3,4,5) or a mutated HSF1 binding site (lane 2). Additionally, supershift experiments with two HSF1 specific antibodies, as well as an IgG antibody as the control, were performed (b). Representative results of four independent experiments in Hep3B cells are shown.
Fig 5: Activation of HSF1 by celastrol diminishes fat accumulation in HepG2 cells. HepG2 cells were stimulated with indicated concentrations of celastrol and cultured in control or PA/OA medium for 96 h. Intracellular fat accumulation was measured by Oil-Red-O staining (a). HepG2 cells were transfected with HSF1 or control siRNA. After 24 h, cells were treated with 0.1 µM celastrol and cultured in control or PA/OA medium for 96 h. Fat accumulation was measured by Oil-Red-O staining (b). The figures depicts the mean values of three independent experiments performed in duplicates ± S.D. * p-values < 0.05.
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