Fig 1: IER5 dephosphorylates HSF1 at multiple Ser and Thr residues including sites involved in repression of HSF1 activity.(A–E) Whole cell extracts and immunoprecipitated samples were analyzed by Western blotting. (A) 293T cells were transfected with HSF1-Flag together with control vector or an IER5 expression vector. Cells were harvested 21 hrs post-transfection. (B) 293T cells were transfected with control vector or HSF1-Flag expression vector, together with control or IER5-targetting siRNAs. Cells were harvested 49 hrs post-transfection. (C) 293T cells were transfected with HSF1-Flag and control vector or IER5. Cells were harvested 27 hrs post-transfection. Cell lysates were immunoprecipitated using anti-Flag antibody, and incubated with or without CIAP for 30 min. Total HSF1 and p-Ser320 were detected. (D) 293T cells were transfected with control vector or HA-HSF1 expression vector. Cells were treated with or without heat shock at 42 °C for 3 hrs, and harvested 24 hrs post-DNA transfection. (E) 293T cells were transfected with HA-HSF1 and control or IER5-Flag expression vector. Cells were harvested 24 hrs post-DNA transfection. (F) HSF1 modification was analyzed by LC-MS/MS. The experiment was performed nine times, and the numbers of analysis that detected each phosphorylation are shown. Significant reductions in phosphorylation at 5 residues (S121, S307, S314, T3232 and T367) were detected. Phosphorylation sites previously reported to be involved in the repression of HSF1 activity (S121 and S307) are shown in blue. (G) 293T cells were transfected with the indicated plasmids and harvested 24 hrs post-transfection. HSPA1A mRNA expression was analyzed by quantitative RT-PCR and other proteins were detected by Western Blotting. (***p < 0.001, #p < 0.0001). (H,I) 293T cells were transfected with control vector or IER5-Flag expression vector. Cells were treated with or without heat shock at 42 °C for 3 hrs, and harvested 24 hrs post-DNA transfection. Endogenous HSF1, IER5-Flag protein expression was analyzed by Western blotting (H), and HSPA1A, HSPA6 and HSPA1B mRNA expression was analyzed by quantitative RT-PCR (I). (*p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001).
Fig 2: 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.
Fig 3: IER5 mRNA expression is increased in various cancers and IER5 is required for anchorage-independent cancer cell proliferation.(A) Mouse embryonic fibroblasts and NIH3T3 cells were treated with TPA (160 ng/ml) for 30 min. NIH3T3 cells were also cultured with 0.5% FBS DMEM medium overnight and then stimulated by 10% FBS DMEM for 2 hrs. Expression of IER5 mRNA was analyzed by quantitative RT-PCR. (*p < 0.05, **p < 0.01). (B) IER5 mRNA expression levels were analyzed in the indicated cancers and their corresponding normal tissues using the Gene Logic SCIANTIS database. (*p < 0.05, ***p < 0.001). (C) The IER5 gene is associated with super-enhancers in various cancer cell lines. The locus of the IER5 gene is shown with both of the human genome reference versions, Hg19/Build37 (upper) and Hg18/Build36 (lower). The regions identified as containing super enhancers by Hnisz et al. are shown by yellow lines17. (D) IER5 mRNA expression in various cancer cell lines. Significance of difference was calculated between non-super enhancer cell lines and super enhancer cell lines. (**p < 0.01, ***p < 0.001, #p < 0.0001). (E–G) OE33 cells were plated in adherent (E) or suspension (F) 96-well culture plates, and control or IER5-targeting siRNAs were introduced. Cell growth assays were performed at the indicated days. Mean relative cell numbers ±SD are shown. IER5 mRNA expression was analyzed by quantitative RT-PCR 48 hrs post-transfection (G). (**p < 0.01, #p < 0.0001). (H–J) Anchorage-independent growth in soft agar was analyzed. Control or IER5-targeting siRNAs were introduced into OE33 cells. The representative images (H) and the mean numbers of colonies ±SD are shown (I). Expression of IER5 (J) was analyzed by quantitative RT-PCR 48 hrs post-transfection. (*p < 0.05, **p < 0.01). (K,L) Cell growth assays were performed using the indicated cell lines. Cells were plated in suspension culture plates and analyzed as in (F). Cell growth was analyzed 7 days (HCC1954, MCF7 and HeLa cells) or 8 days (MDA-MB-231 cells) post-transfection (K). IER5 mRNA expression was analyzed by quantitative RT-PCR 48 hrs post-transfection (L). (*p < 0.05, **p < 0.01).
Fig 4: HSP family genes are induced by IER5.(A) H1299 or 293T cells were transfected with control vector or an IER5 expression vector. Cells were harvested 21 hrs or 27 hrs post-transfection and microarray expression analysis was performed. The table shows the HSP family genes, among the genes induced by IER5. (B) H1299 cells were transfected with control, IER5-Flag or mutant IER5-Flag expression vectors (representative image of mut 1 is shown in Fig. S1). Cells were harvested 27 hrs post-transfection, and mRNA expressions of the HSP family genes were analyzed by Northern blotting. (C) H1299 and 293T cells were transfected with control vector or IER5-Flag expression vector, and cells were harvested 24 hrs post-transfection. Expressions of the HSP family proteins were analyzed by Western blotting. (D–F) Control or IER5-targeting siRNAs were introduced into OE33 cells. Cells were harvested 52 hrs post-transfection. Expression of IER5 (D,F) and HSPA1A (E,F) were analyzed by quantitative RT-PCR (D,E) and Western blotting (F). (**p < 0.01). (G) The promoter regions of HSPA1A, HSPA1B and HSPA6 were inserted into the luciferase reporter plasmid containing a minimal promoter, and assayed 24 hrs post-transfection. Experiments were run in triplicate, and data are represented as the mean-fold activation ±SD. (H) Serially deleted regions of the HSPA1A promoter were analyzed as in (G). Numbers indicate the position of the 5' most nucleotide relative to the transcription initiation site. A heat shock element (HSE), to which HSF1 binds, was found between positions -132 and -109.
Fig 5: IER5 is a novel activator of HSF1.(A–C) Control or HSF1-targeting siRNAs were introduced into H1299 cells. Subsequently, cells were transfected with control vector or an IER5 expression vector 24 hrs post-siRNA transfection. Cells were harvested 24 hrs post-DNA transfection. Expression of HSPA1A (A), HSPA6 (B) and HSF1 (C) mRNAs were analyzed by quantitative RT-PCR. (#p < 0.0001). (D) 293T cells were transfected with HA-HSF1 and control vector or IER5-Flag. Cells were harvested 24 hrs post-transfection. Cell lysates were crosslinked by DSP (1 mg/ml), and immunoprecipitated using anti-HA antibody. HSP90 binding to HA-HSF1 was detected by Western blotting. (E) H1299 cells were transfected with control vector or IER5-Flag expression vector. Cells were harvested 25 hrs post-transfection. Whole cell lysates were crosslinked with EGS. HSF1 and IER-Flag expression was analyzed by Western blotting. (F) H1299 cells were transfected with control vector, IER5-Flag, mut 1-Flag expression vector. Cells were harvested 24 hrs post-transfection. Subcellular localizations of endogenous HSF1 (detected using anti-HSF1 antibody), IER5-Flag and mut 1 (detected using anti-Flag antibody) were analyzed. HSF1 and IER5 expression levels were quantitated and shown at the bottom. (G) HSF1 binding to Heat Shock Elements (HSEs) was analyzed by streptavidin pull-down assay. Biotinylated control or HSE-containing DNA probes were bound to streptavidin-coated beads and mixed with control or IER5-expressing cell lysates. Bead-bound HSF1 was denatured in Laemmli buffer and analyzed by Western blotting.
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