Effect of HSF1 knockdown and mutation on HYPK expression.

<p>A. Gel image representative of three (n = 3) independent experiments for sqRT-PCR of <i>HSF1</i>, <i>HYPK</i> and <i>hsp70</i> expression in HeLa cells transiently expressing empty pSUPER vector, siRNA sequence of human HSF1 cloned in pSUPER vector and scrambled RNA sequence cloned in pSUPER vector. Expression of <i>β-actin</i> was taken as endogenous control. B. Bar graph showing the mean IOD of bands obtained in A. The expression level of <i>HSF1</i>, <i>HYPK</i> and <i>hsp70</i> in each sample was normalized by the corresponding <i>β-actin</i> expression level. Fold change was calculated by considering the relative expression level of <i>HSF1</i>, <i>HYPK</i> and <i>hsp70</i> respectively, in control HeLa cells (expressing empty pSUPER vector) to be 1. C. Western blot analysis for the expression of HSF1, HYPK and Hsp70 in three independent experiments (n = 3) in samples as indicated in A. Expression of β-actin was taken as loading control. D. Bar graph showing the mean IOD of bands obtained in C. The expression level of HSF1, HYPK and Hsp70 in a sample was normalized by the corresponding β-actin band. Fold change was calculated taking the relative expression of HSF1, HYPK and Hsp70 expression in control HeLa cells (expressing empty pSUPER vector) to be 1.E. Gel image representative of three (n = 3) independent experiments for sqRT-PCR of <i>HYPK</i> expression in HeLa cells transiently transfected with empty pSUPER vector, siRNA sequence against human HSF1 cloned in pSUPER vector and scrambled RNA sequence cloned in pSUPER vector, and subjected to HS treatment 72 h after transfection ( HS at 42°C for 60 min followed by recovery at 37°C for 4 h). Expression of <i>β-actin</i> was taken as endogenous control. F. Graph showing the mean IOD of bands obtained in E. The expression level of <i>HYPK</i> in a sample was normalized by the corresponding <i>β-actin</i> band. Fold change was calculated taking the relative expression of <i>HYPK</i> expression in control HeLa cells to be 1. G. Representative gel image of three independent experiments (n = 3) for sqRT-PCR of human <i>HYPK</i> and <i>hsp70</i> gene expression in HeLa cells transiently overexpressing empty pcDNA vector (control), wild type HSF1 (WT HSF1) cloned in pcDNA vector and constitutively active HSF1 (CA HSF1) cloned in pcDNA vector. Expression of <i>β-actin</i> was taken as endogenous control. H. Bar graph showing the mean IOD of bands obtained in G. The expression level of <i>HYPK</i> and <i>hsp70</i> in a sample was normalized by the corresponding <i>β-actin</i> expression level. Fold change was calculated by considering the relative expression level of <i>HYPK</i> and <i>hsp70</i> in control HeLa cells (expressing empty pcDNA vector) to be 1. I. Immunoblot showing expression of HYPK and Hsp70 in three independent experiments (n = 3) in samples as described in G. Expression of β-actin was taken as loading control. J: Bar graph showing the mean IOD of bands obtained for HYPK and Hsp70 in I. The IOD of each HYPK and Hsp70 band was normalized by the corresponding β-actin band. Fold change was calculated taking the relative expression of HYPK and Hsp70 expression in control HeLa cells (expressing empty pcDNA vector) to be 1. <i>Error bars</i> indicate ± SD. The statistical significance level between various experimental pairs is indicated (*,<i>p</i><0.05; **,<i>p</i><0.01; ***,<i>p</i><0.001; NS = not significant).