Fig 2: NMDAR Signaling through GKAP Promotes Invasion in Both Mouse and Human PDAC Cell Lines(A) Left panels: western blot analysis of GKAP and p-GluN2b levels in mPDAC-4361 and mPDAC-2263 cell lines. The numbers indicate quantification (n = 3). Right panel: invasion assay in static and flow-stimulated conditions. Two-way ANOVA, Bonferroni's multiple comparisons test (right panel): ****p < 0.0001 (mean ± SEM, n = 3 invasion assay devices per condition per cell line in one experiment; two independent experiments).(B) MK801 treatment of mPDAC cell lines in static and flow-stimulated invasion assays. The data were normalized to each corresponding “control” in the same static/flow conditions. Two-way ANOVA, Bonferroni's multiple comparisons test: n.s., not significant; *p < 0.05 (mean ± SEM, n = 3 invasion assay devices per condition per cell line in one experiment; two independent experiments).(C and D) GKAP mRNA was knocked-down in mPDAC-4361 (C) and in two hPDAC cell lines, DanG and SUIT2 (D). The knockdown efficiency was assessed by western blot analysis; numbers below indicate levels of GKAP normalized to GAPDH. Cell invasiveness in the invasion assays is shown in bar graphs. Representative images of DAPI-stained nuclei from the invasion assay illustrate the cells that reached the other side of the membrane of a Boyden chamber (scale bar, 100 µm) (C). Two-way ANOVA, Bonferroni's multiple comparisons test (C) or unpaired t test (D): *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. For all invasion assays: n = 3 invasion assay devices per condition in one experiment. Mean ± SEM. Two independent experiments.(E) Western blot analysis of p-HSF1 and FMRP levels in control and MK801-treated hPDAC cells. The numbers below indicate levels of p-HSF1 and FMRP normalized to GAPDH (n = 3).(F) Western blot analysis assessing siRNA-mediated HSF1 and FMRP knockdown in DanG cells and SUIT2 cells are shown in the left panels. The numbers below indicate levels of HSF1 and FMRP normalized to GAPDH. Flow-guided-invasion assays of hPDAC cells after HSF1 or FMRP knockdown are shown in bar graphs on the right). Mean ± SEM, unpaired t test: *p < 0.05; **p < 0.01 (n = 3 invasion assay devices/condition in one experiment). Two independent experiments were performed with consistent results.

Fig 3: HSF1 aptamer inhibits mitogenic signaling.(A) HSF1 inhibition attenuates EGF receptor activation following the addition of EGF to HeLa cells. (B) HSF1 inhibition by iaRNAHSF1 causes a depletion of the total levels and activated forms of Erk1/2. The left most three lanes are a serial dilution of parental line extracts that provides a quantification standard curve. Ectopic expression of HSP70 or HSP90 suppresses the inhibition of mitogenic signaling in the iaRNAHSF1 expressing cells.

Fig 4: The involvement of aberrant HSF1 degradation and HSP70 a5 (BiP/GRP78) attenuation in tau accumulation and toxicity in tauopathy.(A and B) Reduced HSP70 a5 (BiP/GRP78) expression common in human AD brains (A) and cellular and mouse tauopathy models (B). Graphs show reduction in relative HSP70 a5 (BiP/GRP78) protein expression normalized to β-actin in N2a-TauRD ΔK280 (n = 4, * P < 0.05), human AD brains (n = 5, non-significant) and rTg(tauP301L)4510 (n = 5, * P < 0.05). Data represents means ± SEM. Β–actin for Fig 6B was shown in Figs 1B and 5A. (C and D) N2a-TauRD ΔK280 were transfected with either HSF1 WT or HSF1 S303A (constitutively active form) (C) or HSP70 a5 (BiP/GRP78) or BiP siRNA (D). Control N2a-TauRD ΔK280 transfected with empty vector. (C) A right graph on the relative HSP70 a5 (BiP/GRP78) and GFP-TauRD protein expression levels normalized to β-actin in N2a-TauRD overexpressing HSF1 WT (means ± SEM, *P < 0.05, HSP70 a5 (Bip/GRP78); **P < 0.01, GFP- TauRD, comparing to control, n = 4). (D) Reduced GFP-TauRD accumulation by HSP70 a5 (BiP/GRP78) overexpression in N2a-TauRD ΔK280. (E) Cell viability in N2a-TauRD ΔK280 overexpressing either HSF1 WT or HSF1 Δ156–226, or HSP70 a5 (BiP/GRP78) and control was measured by MTT assay (means ± SEM, *P < 0.05, n = 5).

Fig 5: 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).