Journal: Free radical research

Article Title: DCPIP (2,6-dichlorophenolindophenol) as a genotype-directed redox chemotherapeutic targeting NQO1*2 breast carcinoma

doi: 10.3109/10715762.2010.526766

Figure Lengend Snippet: DCPIP induces stress response gene expression and p53-Ser15 activational phosphorylation in MDA-MB231 breast carcinoma cells. (A) The scatter blot (left panel) depicts differential gene expression as detected by the RT2 Human Stress and Toxicity Profiler™ PCR Expression Array technology profiling the expression of 84 (oxidative) stress- and toxicity-related genes after DCPIP treatment (20 μM, 24 h). Upper and lower lines represent the cut-off indicating 4-fold up- or down-regulated expression, respectively. Arrows specify selected genes with at least 4-fold up-regulated expression vs untreated controls. Expression array analysis was performed in three independent repeats and analysed using the two-sided Student’s t-test. The table (right panel) summarizes statistically significant expression changes by at least 3-fold (p < 0.05). (B–G) Immunoblot detection of DCPIP-induced protein levels in MDA-MB231 cells: (B) Modulation of cellular heme oxygenase-1 (HO-1) protein levels by DCPIP (10 and 20 μM, 24 h) as examined by immunoblot analysis of total cellular protein extracts. Detection of α-actin expression served as a loading control. (C) Early time course of DCPIP-modulation (20 μM; 3–12 h) of HO-1 protein levels. (D) Time course of DCPIP-modulation (10 μM; 3–24 h) of Hsp70 and Hsp70B′ protein levels. (E) DCPIP-modulation (10 and 20 μM, 24 h) of cellular p21 protein levels. (F) Time course of DCPIP-modulation (20 μM; 3–24 h) of p21 protein levels. (G) Early activational phosphorylation of p53 as assessed by immunoblot analysis of p-p53 (Ser15) and total p53 protein levels (DCPIP 10–20 μM; 3 h exposure).

Article Snippet: The following primary antibodies were used: rabbit anti-HSP70 polyclonal antibody (SPA-811; 1:1000) and mouse anti-HSP70B′ monclonal antibody (SPA-754; 1: 1000; Assay Designs, Ann Arbor, MI); monoclonal mouse anti-p53 IgG (sc-71817; 1:1000) and polyclonal rabbit anti-phospho-p53 (Ser15) IgG (sc-101762; 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-p21 monoclonal antibody (1:2000) and monoclonal rabbit anti-PARP antibody (1:1000; 46D11, Cell Signaling Technology, Danvers, MA) [ 33 , 35 ].

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MiR-196a regulates heme oxygenase-1 by silencing Bach1 in the neonatal mouse lung

doi: 10.1152/ajplung.00428.2015

Figure Lengend Snippet: Developmental expression of microRNA (miR)-196a, miR-27b, miR-410, mRNA of the transcriptional repressor BTB and CNC homology 1 (Bach1), and proteins of Bach1 and the activator nuclear factor-E2-related factor-2 (Nrf2) in the mouse lung. A: expression levels of miR-196a, Bach1 mRNA, and Bach1 protein during postnatal lung development were measured by real-time PCR (n = 6/time point) and Western blot (n = 5/time point). Levels of miR-196a are normalized to those of small nucleolar RNA 202 (sno202). Bach1 mRNA levels are normalized to those of 18S ribosomal RNA (18S). Values are presented as means ± SE. B: lungs from wild-type (WT) C57BL6 mice during lung development were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using the bincinchoninic acid (BCA) method. Equal concentrations were then loaded onto a gel and blotted for Bach1 and calnexin as a loading control. The Western blots shown are the best representatives of five experiments using five different animals in each condition. C: expression levels of miR-27b, miR-410, and Nrf2 protein during postnatal lung development were measured by real-time PCR (n = 5/time point) and Western blot. Values of miR-27b and miR-410 levels are presented as means ± SE from five mice. Levels of miR-27b and miR-410 are normalized to those of sno202. D: lungs from WT C57BL6 mice during lung development were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using the BCA method. Equal concentrations were then loaded onto a gel and blotted for Nrf2 and calnexin as a loading control (n = 5/time point). Data are shown as a relative level normalized to day 0 neonates. †P < 0.01; *P < 0.05 vs. day 0 for miR-196a, day 0 for Bach1 protein, day 0 for Nrf2 protein, and day 0 for miR-27b and miR-410.

Article Snippet: Protein levels were determined as described ( 29 , 30 ) and were detected using an enhanced chemiluminescence detection kit (GE Healthcare) after overnight incubation of the membrane with the following antibodies: HO-1 (SPA-896, dilution 1:1,000; Stressgen), Bach1 (A1-5, dilution 1:1,000) ( 34 ), calnexin (SPA-860, dilution 1:10,000; Santa Cruz Biotechnology), and Nrf2 (sc-722 dilution 1:500; Santa Cruz Biotechnology), followed by secondary anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (dilution 1:5,000; Santa Cruz Biotechnology).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Protein Concentration, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MiR-196a regulates heme oxygenase-1 by silencing Bach1 in the neonatal mouse lung

doi: 10.1152/ajplung.00428.2015

Figure Lengend Snippet: Differential regulation of lung miR-196a, Bach-1, and heme oxygenase-1 (HO-1) in neonate and adult mice exposed to hyperoxia. A–C: miR-196a, HO-1 mRNA, and Bach1 mRNA levels, respectively, in neonatal and adult lungs exposed to hyperoxia. Values are presented as means ± SE of six separate determinations. D: Bach1 protein level in neonatal and adult lungs exposed to hyperoxia. Calnexin (CNX) is shown as a loading control. Densitometric evaluation of protein expression normalized to calnexin is provided. Values are presented as means ± SE of four separate determinations; *P < 0.05 vs. air.

Article Snippet: Protein levels were determined as described ( 29 , 30 ) and were detected using an enhanced chemiluminescence detection kit (GE Healthcare) after overnight incubation of the membrane with the following antibodies: HO-1 (SPA-896, dilution 1:1,000; Stressgen), Bach1 (A1-5, dilution 1:1,000) ( 34 ), calnexin (SPA-860, dilution 1:10,000; Santa Cruz Biotechnology), and Nrf2 (sc-722 dilution 1:500; Santa Cruz Biotechnology), followed by secondary anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (dilution 1:5,000; Santa Cruz Biotechnology).

Techniques: Control, Expressing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MiR-196a regulates heme oxygenase-1 by silencing Bach1 in the neonatal mouse lung

doi: 10.1152/ajplung.00428.2015

Figure Lengend Snippet: Hyperoxia alters miR-196a and Bach-1 in mouse embryonic fibroblast cells (MEFs). A: luciferase activity after hyperoxia in MEFs. Twenty-four hours after transfection of the Bach1 3′-untranslated region (UTR) plasmid, MEFs were exposed to hyperoxia. Relative light intensity was measured 4, 8, and 24 h after hyperoxia. The color scale bar represents photon emission intensity. Values are presented as means ± SE of six separate determinations; *P < 0.05. B: miR-196a levels were measured by qPCR. Levels of miR-196a are normalized to those of sno202. C: Bach1 protein levels were measured by Western blot [calnexin (CNX) is shown as a loading control], and densitometric evaluation of protein expression normalized to loading control (CNX). D and E: HO-1 mRNA and Bach1 mRNA levels were measured by qPCR. Levels of miR-196a are normalized to those of sno202; HO-1 and Bach1 mRNA levels are normalized to those of 18S. Values are presented as means ± SE of four separate determinations; *P < 0.05.

Article Snippet: Protein levels were determined as described ( 29 , 30 ) and were detected using an enhanced chemiluminescence detection kit (GE Healthcare) after overnight incubation of the membrane with the following antibodies: HO-1 (SPA-896, dilution 1:1,000; Stressgen), Bach1 (A1-5, dilution 1:1,000) ( 34 ), calnexin (SPA-860, dilution 1:10,000; Santa Cruz Biotechnology), and Nrf2 (sc-722 dilution 1:500; Santa Cruz Biotechnology), followed by secondary anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (dilution 1:5,000; Santa Cruz Biotechnology).

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Control, Expressing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MiR-196a regulates heme oxygenase-1 by silencing Bach1 in the neonatal mouse lung

doi: 10.1152/ajplung.00428.2015

Figure Lengend Snippet: MicroRNA-196a regulates Bach1 and HO-1 expression. A–C: luciferase activity was determined by measuring photon emission after incubation with the substrate luciferin for 24 h after transfection of Bach1 3′-UTR plasmid and miR-196a control, mimics, and inhibitor. Values for cells with control transfection are set equal to 1. Data are presented as means ± SE of six to eight separate determinations. D and E: effect of miR-196a mimic (30 nM) or miR-196a inhibitor (10 nM) on miR-196a and Bach1 levels. MEFs were transfected with miRNA-196a mimics and inhibitor with Lipofectamine 2000 for 72 h. miR-196a levels and Bach1 mRNA levels were measured by qPCR and compared with mock transfection (mock transfection represents mice treated with Lipofectamine alone). Levels of miR-196a are normalized to those of sno202. Bach1 mRNA levels are normalized to those of 18S. Values are presented as means ± SE of three separate determinations; *P < 0.05 vs. mock transfection. F and G: Bach1 protein levels after transfection with miR-196a mimic and inhibitor were measured by Western blot. Calnexin is shown as a loading control. Densitometric evaluation of protein expression normalized to loading control (calnexin; CNX) is provided. Values are presented as means ± SE of three separate determinations; *P < 0.05 vs. 0 nM, **P < 0.001 vs. 0 nM. H: effect of miR-196a mimic (30 nM) or miR-196a inhibitor (10 nM) on HO-1 mRNA levels. MEFs were transfected with miRNA-196a mimics and inhibitor with Lipofectamine 2000 for 72 h. HO-1 mRNA levels were measured by qPCR and compared with mock transfection. HO-1 mRNA levels are normalized to those of 18S. Values are presented as means ± SE of three separate determinations; *P < 0.05 vs. mock transfection.

Article Snippet: Protein levels were determined as described ( 29 , 30 ) and were detected using an enhanced chemiluminescence detection kit (GE Healthcare) after overnight incubation of the membrane with the following antibodies: HO-1 (SPA-896, dilution 1:1,000; Stressgen), Bach1 (A1-5, dilution 1:1,000) ( 34 ), calnexin (SPA-860, dilution 1:10,000; Santa Cruz Biotechnology), and Nrf2 (sc-722 dilution 1:500; Santa Cruz Biotechnology), followed by secondary anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (dilution 1:5,000; Santa Cruz Biotechnology).

Techniques: Expressing, Luciferase, Activity Assay, Incubation, Transfection, Plasmid Preparation, Control, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MiR-196a regulates heme oxygenase-1 by silencing Bach1 in the neonatal mouse lung

doi: 10.1152/ajplung.00428.2015

Figure Lengend Snippet: miR-196a and HO-1 expression after transfection of miR-196a mimic and inhibitor in Bach1 KO MEFs. A: miR-196a levels after transfection of miR-196a mimic and inhibitor. Values are presented as means ± SE (n = 3); *P < 0.05 vs. mock transfection. B: HO-1 mRNA levels after transfection of miR-196a mimic and inhibitor. Levels of miR-196a are normalized to those of sno202, HO-1 mRNA levels are normalized to those of 18S. Values are presented as means ± SE (n = 3); *P < 0.05 vs. mock transfection. C and D: Western blots and analysis of Bach1 in Bach1 KO and WT MEFs and HO-1 protein after transfection of miR-196a mimic and inhibitor. HO-1 protein levels are normalized to those of calnexin. Values are presented as means ± SE (n = 3); *P < 0.05 vs. mock transfection.

Article Snippet: Protein levels were determined as described ( 29 , 30 ) and were detected using an enhanced chemiluminescence detection kit (GE Healthcare) after overnight incubation of the membrane with the following antibodies: HO-1 (SPA-896, dilution 1:1,000; Stressgen), Bach1 (A1-5, dilution 1:1,000) ( 34 ), calnexin (SPA-860, dilution 1:10,000; Santa Cruz Biotechnology), and Nrf2 (sc-722 dilution 1:500; Santa Cruz Biotechnology), followed by secondary anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (dilution 1:5,000; Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Western Blot

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( 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 ). 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.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Transfection, Plasmid Preparation, Expressing, Microarray, Mutagenesis, Northern Blot, Western Blot, Quantitative RT-PCR, Luciferase, Activation Assay

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( 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.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Immunoprecipitation, Binding Assay, Western Blot, Pull Down Assay

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( 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).

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Expressing, Incubation, Modification, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( 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.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Immunoprecipitation

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A–D ) OE33 cells (2 × 10 3 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.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Stable Transfection, Western Blot