Figure 6
From: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis
IER5 induces PP2A-dependent dephosphorylation and activation of HSF1.
(A–C) 293T cells were transfected with HA-HSF1 together with control vector or IER5-Flag. Cells were treated with 20 mM NaF acid for 4 hrs and harvested 24 hrs post-transfection. Expression of HSPA1A and HSPA6 were analyzed by quantitative RT-PCR (A,B) and expression of IER5-Flag and HA-HSF1 were detected by Western Blotting (C). (**p < 0.01, ***p < 0.001). (D) 293T cells were transfected with HA-HSF1 together with control vector or IER5-Flag. Cells were treated with 500 nM okadaic acid for 8 hrs and harvested 24 hrs post-transfection. Expression of HSPA1A was analyzed by quantitative RT-PCR and expression of IER5-Flag and HA-HSF1 were detected by Western Blotting. (***p < 0.001). (E) 293T cells were transfected as in (D). Cells were treated with 250 nM okadaic acid for 4 hrs and harvested 24 hrs post-transfection. Cell lysates were immunoprecipitated using anti-HA antibody. Total HSF1 and HSF1 phosphorylated at Ser121 or p-Ser303, 307 were detected by Western Blotting. (F–H) Control or PPP2CA (PP2A catalytic subunit)-targeting siRNAs were introduced into 293T cells. Subsequently, cells were transfected with HA-HSF1 and control vector or IER5-Flag expression vector 72 hrs post-siRNA transfection. Cells were harvested 24 hrs post-DNA transfection. IER5, HSF1 and PPP2CA protein levels were analyzed by Western blotting and HSPA1A (G) and HSPA6 (H) mRNAs were analyzed by quantitative RT-PCR. (**p < 0.01). (I,J) 293T cells were transfected with HA-HSF1 together with control vector, IER5-Flag or IER5-Flag mut 1-Flag and harvested 24 hrs post-transfection. IER5-Flag (I) or HA-HSF1 (J) was immunoprecipitated using anti-Flag or anti-HA antibodies. Association of HSF1, PP2A catalytic subunit PPP2CA and PP2A regulatory subunit B55 with IER5 was analyzed by Western blotting in (I) and association of IER5, PPP2CA and B55 with HSF1 was analyzed in (J). (K) Endogenous IER5 in OE33 cells was immunoprecipitated using anti-IER5 antibody. Association of HSF1 and PPP2CA with IER5 was analyzed. (L) PP2A phosphatase activities were analyzed using the indicated amounts of cell lysates. 293T cells were treated with or without Okadaic acid (500 nM) for 8 hrs. Control or IER5-Flag expression vector were transfected and harvested 24 hrs post-transfection.
