Figure 3

Heat shock factor is responsible for Hsp16 elevation and heat shock-mediated suppression of the Vpr activities. (A) Overexpression of hsf1 suppressed Vpr-induced G2 arrest. a, overexpression of hsf1 reduced Vpr’-induced cell elongation back to normal size. Cell images were captured 30 hrs after gene induction. b, overexpression of vpr or vpr’ suppressed Vpr-induced G2 arrest as measured by flow cytometric analysis. The vpr-repressing (off) and vpr-expressing (on) cells were prepared as described previously [37]. Forty-eight hours after vpr gene induction, cells were collected for flow cytometric analysis. (B) No additional reduction of Vpr-induced cell elongation was seen when hsf1-expressing cells were treated with high temperature (36°C). Average and standard deviation of cell length was calculated based on three independent experiments by counting a minimum 90 cells. (C) Inducible expression of HIV-1 vpr’ and constitutive expression of hsf1 under normal (30°C) and high (36°C) temperature shown by Western blot analysis. Proteins were extracted from cells cultured 30 hrs after gene induction (GI). Because of the lack of an antibody against fission yeast anti-Hsf1 and our interest in monitoring Hsf1-mediated Hsp16 elevation, Hsp16 protein production was used here as marker for Hsf1 activity [35].