Fig 2: HSF1 inhibition attenuates cancer cell survival.(A) iaRNAHSF1 expression does not affect normal cells (IMR90), yet induces an abnormal morphology in HeLa cervical carcinoma and chemically transformed kidney cells (293T). (B). Nuclear condensation and fragmentation assays reveal that iaRNAHSF1 expression induces ~10-fold increase in apoptosis in HeLa cells (p<0.0001) (n = 8), and ~7-fold increase in apoptosis in 293T cells (p<0.0001) (n>8). Furthermore, iaRNAHSF1 induced apoptosis in effectively suppressed by the over-expression of molecular chaperones (HSF1 p<0.006, Hsp90 p<0.005, or Hsp70 p<0.002), but not random proteins (GFP or LacZ) (n>8).

Fig 3: Early sex-specific responses to poly I:C in the brain of P8 mice. Increased caspase-3 (A) and caspase-8 activity (B), IRF3 nuclear translocation (D), and INF-ß gene upregulation (F) occur 6 h after poly I:C in the brain of female mice. Increased NF?B (C) and IRF3 (D) nuclear translocation, GFAP expression (E) and INF-ß gene upregulation (F) occur 14 h after poly I:C in the brain of male mice. Data are presented using box and whiskers plots, where boxes are limited by the 75th and 25th percentiles interquartile range (IQR)] and whiskers are limited by the last data point within 1.5 times the IQR from the median (continuous line inside the box). (A–E) White boxes represent NS-treated pups, while gray boxes represent poly I:C-treated pups. (D) Representative immunoblots showing translocation of IRF3 and AP1 from the cytosolic fraction (CF) to the nuclear fraction (NF) in males and females at 6 and 14 h after poly I:C. Top immunoblots show purity of the NF and CF using Lamin B1 and HSP90, respectively. (E) Representative immunoblots showing GFAP expression in cytosolic fractions. (F) Shows the fold change in INF-ß mRNA levels in the brain of male and female mice at 6, 14, 24 h and 7 days after poly I:C relative to NS-treated mice (discontinuous line sitting at 1). °, outlier (between 1.5 and 3 times the IQR); •, extreme (> 3 times the IQR); *p < 0.05.

Fig 4: (A,B) Western blot analysis of LC3II (A) and p62 (B) proteins extracted from HeLa cells left untreated (-) or treated (+) with 250 µM of CoCl2 alone or in combination with 20 µM of chloroquine. Proteins were normalized using HSP90; below the bar graph are shown the representatives Western blot scan. Bar graphs represent means ± SD (N = 3), two-way ANOVA (Tukey’s multiple comparison test). *p value < 0.05 and **p value < 0.01. (C–F) Western blot analysis of LC3II (C), p62 (D), and COXII (E) and COXIV (F) proteins extracted from HeLa cells left untreated (-) or treated (+) with 250 µM of CoCl2 alone or in combination with 1 mM of CoCl2 at different exposure time 4, 16, and 24 h. Proteins were normalized using HSP90, and below the bar graph (C–E) are shown the representatives Western blot scan. Bar graph represents means ± SD (N = 3), two-way ANOVA (Tukey’s multiple comparison test). *p value < 0.05, **p value < 0.01, ***p value < 0.001, and ****p value < 0.0001.

Fig 5: Effective targeting of HSF1 activity reduces the levels of molecular chaperone proteins.(A) Western blot analysis showing the depletion of specific molecular chaperone proteins in aptamer or control expressing cells. Hsp90 co-expression rescues specific molecular chaperones observed in HSF1 inhibited aptamer expressing cells. The asterisk indicates PARP degradation product, a marker of apoptosis. The left most three lanes are a serial dilution of parental line extracts that provides a quantification standard curve. (B) Quantification of the results observed in panel A (n = 4, error indicates %SEM).