HSF1 knockdown reduces colony formation.

<p>SKOV3.shHSF1B, HEY.shHSF1B and control cell lines were plated 250 cells per well in 6-well plates in triplicate. Cell were treated with or without 1 μg/ml doxycycline (Dox) to induce HSF1 knockdown and were given an additional dose after 4 days. Cells were stained with crystal violet after 8 days to visualize colonies.</p

HSF1 levels are elevated in ovarian cancer patient samples.

<p>A, HSF1 copy number is increased most frequently in ovarian cancer. HSF1 copy number was analyzed in a variety of cancers using TCGA data and GISTIC analysis with a threshold CNA change of +/-2. B, HSF1 transcripts are elevated in a variety of cancers. Samples from tumor tissue and matched normal tissue were compared in the TCGA database using RNA Seq V2 RSEM data with a z-score threshold of +/-2. C, HSF1 is increased at the mRNA level in an ovarian cancer data set GSE18520 consisting of 10 normal ovarian samples and 53 late stage, primary site, high grade ovarian cancer samples. D, HSF1 is increased at the mRNA level in a TCGA ovarian cancer data set consisting of 8 normal ovarian samples and 568 ovarian cancer samples.</p

Validation of inducible HSF1 knockdown ovarian cancer cell lines.

<p>A, The heat shock response in the epithelial ovarian carcinoma cell lines SKOV3 and HEY as compared to normal ovarian epithelial T80 cells. T80, SKOV3, and HEY cells were treated with a 42°C heat shock for the indicated times and harvested immediately after. Cell lysates were subjected to Western blot analysis using antibodies recognizing HSF1, HSF1 phosphorylated at S326, HSP90, HSP70, and actin as a loading control. B, The pTRIPZ system was used to create the doxycycline-inducible HSF1 knockdown cell lines SKOV3.shHSF1A, SKOV3.shHSF1B, HEY.shHSF1A and HEY.shHSF1B. After treatment with 1 μg/ml doxycycline for 48 hours, cell lysates were subjected to Western blot analysis using antibodies recognizing HSF1 and actin as a loading control. Both short and long exposures are shown for the HSF1 blot. C, HSF1 knockdown does not cause a large decrease in cell viability. The viability of the SKOV3.shHSF1B and HEY.shHSF1B cell lines as compared to shControl cells was assessed after treatment with 1 μg/ml doxycycline for the indicated times using the PrestoBlue cell viability assay. Mean percent viability (n = 8) and standard error is shown.</p

HSF1 knockdown inhibits wound healing, migration and induction of fibronectin.

<p>A, HSF1 knockdown reduces wound closure. Cells treated with or without 1 μg/ml doxycycline were grown in 6-well plates to confluency. Cells were scraped to create wounds, the cells were washed and serum-free media was added. The intersections of perpendicular scratches were photographed immediately and 12 hours after and analyzed using Tscratch software. Asterisk denotes significant difference from all other samples calculated by ANOVA (P <0.05). B, HSF1 knockdown reduces migration. After treatment with or without 1 μg/ml doxycycline and 12 hours of serum starvation, cells were added to a Boyden chamber at 2.5 x 10⁴ cells per chamber. Serum was used as the chemoattractant in the lower chamber. After 16 hours, nonmigrating cells were scrubbed and cells which had migrated stained. The experiment was done in triplicate and analysis done by paired t-test. Asterisk marks significant difference (P < 0.05). C, HSF1 KD reduces TGFβ-induced expression of fibronectin. SKOV3.shHSF1B and HEY.shHSF1B were treated with 1ug/ml doxycycline, 10 ng/μl TGFβ, or both, and cell lysates were harvested for immunoblotting. Cell lysates were subjected to Western blot analysis using antibodies recognizing fibronectin, HSF1, and actin as a loading control.</p

Fibronectin expression is induced by 3D growth.

<p>SKOV3 and HEY cells were cultured under 2D or 3D conditions, with or without TGFβ, as indicated. Cell lysates were subjected to Western blot analysis using antibodies recognizing fibronectin, and actin as a loading control.</p