Figure 2
Overexpression of HSP27 suppresses Vpr-induced cell cycle G2 arrest. (A) Suppression of Vpr-induced G2 arrest by HSP27 in Vpr-transfected HEK293-632 cells. Both the vpr and HSP27 genes were induced by muristerone A as described previously (18) through pZY1-vpr or pZH1-HSP27plasmids, respectively. The extent of Vpr-induced G2 arrest, measured by flow cytometric analysis, was quantified 72 h after gene induction, by the relative G2 to G1 ratio between gene-repressing and gene-expressing cells. A relative G2/G1 ratio close to one indicates no significant difference between the gene-on and gene-off cultures. A relative ratio > 1 suggests increased proportion of G2 cells in the gene-on culture (18). A representative experiment out of three performed is shown; the following P values have been calculated: (pZY + pZH) vs. (Vpr + pZH), P < 0.05; (Vpr + HSP27) vs. (Vpr + pZH), P < 0.05; (pZY + pZH) vs. (Vpr + HSP27), P = 0.31. (B) Western blot analysis of Vpr and HSP27 on an aliquot of cells collected for flow cytometric analysis in panel A. (C) VSV-G pseudotyped Vpr(+) or Vpr(−) HIV-1NLHX (22) was used to infect HSP27-expressing cells. Cell cycle distribution was measured by flow cytometry 48 h after viral infection. Note that this experiment was performed together with analysis of Hsp16 activity, which was published previously (13); therefore, the control panels are the same as shown in that paper.