Figure 7
From: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis
IER5/HSF1/HSP family gene axis in cancer and in stressed cells.
(A–D) OE33 cells (2 × 103 cells) were plated in adherent (A) or suspension (B) 96 well culture plates and control, IER5-targeting or HSF1-targeting siRNAs were introduced. Cell growth assays were performed on the indicated days. Relative cell numbers were analyzed with CellTiter-Glo reagents from four wells and the mean cell numbers ±SD are shown. IER5 and HSF1 mRNA expression was analyzed by quantitative RT-PCR 48 hrs post-transfection (C,D). (**p < 0.01, #p < 0.0001). (E) OE33 cells were stably transfected with caHSF1 and the indicated siRNAs were introduced. Cell lysates were prepared 48 hrs post-transfection and expression of caHSF1-Flag, IER5, HSPA1A/1B were analyzed by Western blotting. (F) Cell growth assays were performed as in (B) using OE33 cells expressing caHSF1. (**p < 0.01). (G,H) Expression of IER5 (G) and HSPA6 (H) and prognosis in cancer patients. Disease-specific survival of patients with bladder cancer (Transitional cell carcinoma, dataset GSE13507) was analyzed using the PrognoScan database. (I–K) Expression of IER5 (I), HSPA6 (J) and HSPA1A (K) mRNA in cells treated with Adriamycin. Cell lines carrying wild-type p53 were treated with Adriamycin (1 μM) for 24 hrs (MRC5, MCF7, U2OS) or 19 hrs (A549). (*p < 0.05, **p < 0.01). (L) Expression of IER5, p53 and HSPA1A protein level in U2OS cells. Control, p53 and IER5 targeting siRNAs were introduced. Cells were treated with Adriamycin (1 μM) for 24 hrs and harvested 48 hrs post siRNA-transfection. (M) IER5 is transiently induced downstream of p53 and activates HSF1 in stressed cells, while IER5 is overexpressed and constitutively activates HSF1 in cancer cells.
