Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: The Egr-1 gene is rapidly induced after TCR-mediated activation of the DPK double positive cell line. ( A ) Total RNA isolated from DPK cells cultured with immobilized anti-CD3ε mAb for the indicated times (shown in hours), was subjected to RT-PCR analysis using Egr-1 or CD4 primers. ( B ) Electrophoretic mobility shift assay using nuclear lysates prepared from DPK cells 8 h after activation by immobilized antiCD3ε mAb. Probes contained a single Egr-1 binding site ( left ) or overlapping Egr-1 and Sp1 sites ( right ). ( C ) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 1 μm pigeon cytochrome c peptide for the indicated times. Total RNA was isolated and subjected to a competitive RT-PCR assay (see Materials and Methods). Note the different scales for Egr-1 and CD4 mRNA expression.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Activation Assay, Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Binding Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: DPK cell differentiation and Egr-2,3 mRNA induction is cyclosporin A sensitive, while Egr-1 mRNA induction is cyclosporin A resistant. ( A , B ) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 2 μM pigeon cytochrome c peptide in the presence or absence of 100 ng/ml cyclosporin A, or appropriate dilution of solvent (DMSO) as indicated. Cells were harvested and stained for CD69 after 1 or 3 d in culture ( A ) or stained for CD4 and CD8 after 3 d in culture ( B ). ( C ) RT-PCR analysis of total RNA derived from DPK cells activated for 6 h with immobilized anti-CD3ε mAb in the presence or absence of 300 ng/ml cyclosporin A, using Egr-1, Egr-2 (Krox-20), CD4, CD69 or Egr-3 primers. (*) Also shown for the indicated samples is the relative level of Egr-1 cDNA normalized to expression of CD4 cDNA as determined by competitive RT-PCR assay. The identity of the lower major band in Egr-3 RT-PCR was verified by sequencing.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Cell Differentiation, Cell Culture, Staining, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr gene family is dependent upon ras signaling pathways. ( A ) Competitive RT-PCR was used to compare expression of Egr-1 and CD4 genes in DPK or 17N4 cells activated for 6 h with immobilized anti-CD3ε mAb. ( B ) RT-PCR analysis of total RNA derived from DPK or 17N4 cells activated for 6 h with immobilized anti-CD3ε mAb. Independent PCR reactions using Egr-2 (Krox-20), CD4, or Egr-3 primers were performed.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Egr-1 mRNA and DNA binding activity in the thymus is MHC dependent. ( A ) Thymocytes derived from wild-type or MHCdeficient mice were two color-stained for CD4 and CD8. ( B ) Competitive RT-PCR was used to determine the level of expression of CD4 and Egr-1 genes in thymocytes derived from wild-type or MHC-deficient mice. ( C ) Electrophoretic mobility shift assay using nuclear lysates prepared from freshly isolated thymocytes derived from wild-type or MHCdeficient mice using a probe containing an Egr-1 binding site. Nuclear extracts derived from 5 × 10 5 cells containing equivalent amounts of protein were used in binding reactions. In some instances as indicated, binding reactions contained anti-Egr-1 antibody or normal rabbit serum (NRS). For comparison, a binding reaction containing recombinant Egr-1 is shown.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Binding Assay, Activity Assay, Derivative Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Electrophoretic Mobility Shift Assay, Isolation, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 mRNA in double positive thymocytes. ( A ) Total thymocytes and CD4 + 8 + thymocytes (isolated by cell sorting, 95% DP) derived from the same animal, were assayed for expression of Egr and CD4 mRNA by competitive RT-PCR. Shown is the relative level of Egr-1 cDNA in the sample, normalized to the level of CD4 cDNA. ( B ) Total thymocytes derived from an MHC-deficient mouse were cultured with hamster immunoglobulin-coated or anti-CD3ε mAbcoated beads for 90 min before determination of Egr-1 and CD4 gene expression as in Fig. C .

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Isolation, FACS, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 protein in the thymus. Thin sections of normal thymus ( A–C ) or MHC knockout thymus ( D ) were fixed in formaldehyde and stained with a specific rabbit anti-Egr-1 peptide antiserum ( A , C , D ) or the same antibody preincubated with specific peptide ( B ). Regions of cortex ( C ) and medulla ( M ) are indicated. Sections were counterstained with hematoxylin and photographed at ×20 ( A , B ) or ×40 ( C , D ). C shows a magnification of the same section photographed in A .

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Knock-Out, Staining

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 protein in thymocyte subsets. Thymocytes from a young adult mouse were 4-color stained for expression of CD4, CD8, Egr-1, and CD69 or CD3, and analyzed by FACS ® as described in Materials and Methods. Indicated in the dot plots are the percentage of thymocytes within each quadrant, or in parenthesis ( upper right dot plot ), the percentage of Egr-1 + thymocytes within each thymocyte subset. Where indicated, staining is shown for gated populations of thymocytes (either Egr-1 + thymocytes as shown in histogram, or CD4 + 8 + thymocytes). Similar results were obtained from three other individual animals.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E 2 synthase up-regulation for colorectal cancer progression

doi:

Figure Lengend Snippet: A. mPGES1 expression is reduced by aspirin (ASP, 1 μM) or etoricoxib (ETOR, 1 μM) treatment for 24 h, in both HT29-LucD6 EV and COX2 cells as quantified by qRT-PCR. B. PTGES mRNA is increased by PGF 2α (1 μM) in EV cells while it is reduced by AL8810 (10 μM) treatment (24 h) in HT29-LucD6-COX2. C, D. Relative luciferase activity of cells transfected with the “full length” (PTGES-A, -631 to -1) and “proximal” (PTGES-B, -177 to-1) constructs of the human PTGES promoter transfected in HT29-EV and –COX2 cells. E. mRNA levels of EGR1 were quantified by RT-PCR in samples from EV and COX2-overexpressing HT29-LucD6 and SW620 cells. F. Relative luciferase activity of an EGR1 reporter construct EV and COX2 in HT29 and SW480ADH cells. EGR1 mRNA G. and protein H. induction after 24 h treatment of the indicated cell lines with 1 μM of the PGs shown. * p < 0,005

Article Snippet: Membranes were incubated with specific antibodies against COX2, mPGES1 (Cayman Chemicals Europe) or anti- EGR1 (sc-189) or anti-HSP90 (sc-7947) antibody (Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E 2 synthase up-regulation for colorectal cancer progression

doi:

Figure Lengend Snippet: A. Relative levels of PTGS2 , EGR1 and PTGES mRNA, estimated by RT-PCR analysis of HT29-EV and –COX2 cells transfected with 3 different shRNA expressing plasmids, specific for EGR1 or one shRNA without any known mammalian target (scr). B. Immunofluorescence for mPGES1 and EGR1 of HT29-EV and –COX2 cells transfected as mentioned above. C. Fluorescence quantification averages of the images of three independed experiments, as in (B) * p < 0,001. Bar: 32 μm.

Article Snippet: Membranes were incubated with specific antibodies against COX2, mPGES1 (Cayman Chemicals Europe) or anti- EGR1 (sc-189) or anti-HSP90 (sc-7947) antibody (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA, Expressing, Immunofluorescence, Fluorescence

Journal: Oncotarget

Article Title: Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E 2 synthase up-regulation for colorectal cancer progression

doi:

Figure Lengend Snippet: Pearson's correlation coefficient calculated from the gene expression data for the genes shown in the two colorectal tumor collections (Gaedcke colorectal and Skrzypczak Colorectal 2; Oncomine)

Article Snippet: Membranes were incubated with specific antibodies against COX2, mPGES1 (Cayman Chemicals Europe) or anti- EGR1 (sc-189) or anti-HSP90 (sc-7947) antibody (Santa Cruz Biotechnology).

Techniques: Gene Expression

Journal:

Article Title:

doi:

Figure Lengend Snippet: Overexpression of HMOX1, ATF3, and EGR1 in HUVECs after treatment with 20.0 and 40.0 μ g/mL of EENL for 24 hours. Protein levels were measured with specific antibodies by western blot analysis; GAPDH was the loading control. Vehicle-treated cells were used as control. The experiments were repeated in triplicates, and the representative blot was shown.

Article Snippet: Proteins were extracted from cells in modified RIPA buffer and western blotting was performed using primary antibodies against heme oxygenase-1 (HMOX1), activating transcription factor 3 (ATF3), and early growth response-1 (EGR1), (from Abcam Inc., Cambridge, MA, USA) and horseradish peroxidase-conjugated secondary antibodies as described previously [ ].

Techniques: Over Expression, Western Blot

Journal:

Article Title:

doi:

Figure Lengend Snippet: (a)

Article Snippet: Proteins were extracted from cells in modified RIPA buffer and western blotting was performed using primary antibodies against heme oxygenase-1 (HMOX1), activating transcription factor 3 (ATF3), and early growth response-1 (EGR1), (from Abcam Inc., Cambridge, MA, USA) and horseradish peroxidase-conjugated secondary antibodies as described previously [ ].

Techniques: Activity Assay, Protein Binding, Binding Assay

Journal: Cancer Medicine

Article Title: SNGH16 regulates cell autophagy to promote Sorafenib Resistance through suppressing miR‐23b‐3p via sponging EGR1 in hepatocellular carcinoma

doi: 10.1002/cam4.3020

Figure Lengend Snippet: Primers sequence used for qPCR

Article Snippet: 40 μg protein samples were separated on 10% SDS‐PAGE and then electrotransferred onto PVDF membrane, which was blocked by 5% skimmed milk at room temperature for 1 hour Next, the membrane was incubated with the primary antibodies LC3I (GTX17380, 1:1000; GeneTex) and LC3II (GTX127375, 1:3000; GeneTex), EGR1 (ab133695, 1:1000; Abcam) at 4°C overnight.

Techniques: Sequencing

Journal: Cancer Medicine

Article Title: SNGH16 regulates cell autophagy to promote Sorafenib Resistance through suppressing miR‐23b‐3p via sponging EGR1 in hepatocellular carcinoma

doi: 10.1002/cam4.3020

Figure Lengend Snippet: Prediction and verification of miR‐23b‐3p target gene and the expression of EGR1 in Hep3B or Hep3B/So cells. A, Prediction of miR‐23b‐3p target gene. B, Verification of miR‐23b‐3p target gene detected by dual‐luciferase reporter assay. * P < .05, ** P < .001, vs Mimic. C‐E, EGR1 protein and gene expression in Hep3B and Hep3B/So cells determined by qRT‐PCR, GAPDH served as internal reference. * P < .05, ** P < .001, vs Hep3B. F‐H, EGR1 protein and gene expressions in Hep3B/So cells detected by qRT‐PCR, with the group divided into control, NC, si‐SNHG16, si‐SNHG16 + inhibitor and inhibitor, GAPDH served as internal reference. ** P < .001, vs control, ## P < .001, vs NC, ^^ P < .001, vs si‐SNHG16, △△ P < .001, vs Inhibitor

Article Snippet: 40 μg protein samples were separated on 10% SDS‐PAGE and then electrotransferred onto PVDF membrane, which was blocked by 5% skimmed milk at room temperature for 1 hour Next, the membrane was incubated with the primary antibodies LC3I (GTX17380, 1:1000; GeneTex) and LC3II (GTX127375, 1:3000; GeneTex), EGR1 (ab133695, 1:1000; Abcam) at 4°C overnight.

Techniques: Expressing, Luciferase, Reporter Assay, Quantitative RT-PCR

Journal: Cancer Medicine

Article Title: SNGH16 regulates cell autophagy to promote Sorafenib Resistance through suppressing miR‐23b‐3p via sponging EGR1 in hepatocellular carcinoma

doi: 10.1002/cam4.3020

Figure Lengend Snippet: Overexpression of EGR1 partially reversed the effects of overexpression of miR‐23b‐3p on cell viability, apoptosis and autophagy sorafenib‐resistant Hep3B cells. A, CCK‐8 assay was used to detect the viability of sorafenib‐resistant Hep3B cells. B and C, Flow cytometry was used to detect the apoptosis of sorafenib‐resistant Hep3B cells. D‐F, Western blot was used to detect the expression of LC3I and LC3II in sorafenib‐resistant Hep3B cells. * P < .05, ** P < .001, vs control, # P < .05, ## P < .001, vs mimic control, ^ P < .05, ^^ P < .001, vs mimic, △ P < .05, △△ P < .001, vs EGR1, ‡ P < .05, ‡‡ P < .001, vs NC

Article Snippet: 40 μg protein samples were separated on 10% SDS‐PAGE and then electrotransferred onto PVDF membrane, which was blocked by 5% skimmed milk at room temperature for 1 hour Next, the membrane was incubated with the primary antibodies LC3I (GTX17380, 1:1000; GeneTex) and LC3II (GTX127375, 1:3000; GeneTex), EGR1 (ab133695, 1:1000; Abcam) at 4°C overnight.

Techniques: Over Expression, CCK-8 Assay, Flow Cytometry, Western Blot, Expressing

Journal: Cancer Medicine

Article Title: SNGH16 regulates cell autophagy to promote Sorafenib Resistance through suppressing miR‐23b‐3p via sponging EGR1 in hepatocellular carcinoma

doi: 10.1002/cam4.3020

Figure Lengend Snippet: Tumorigenesis in mice injected with Hep3B or Hep3B/So cells and the expressions of EGR1 and LC3II and LC3I, with group divided into Hep3B, Hep3B/So, Hep3B + So, Hep3B/So + So, Hep3B/So + si‐SNHG16 + inhibitor+So, Hep3B/So + si‐SNHG16 + inhibitor+So, Hep3B/So + inhibitor+So. A and B, Tumor volume from scarified mice. C‐E, EGR gene and protein expression levels detected by qRT‐PCR and western blot. F‐H, LC3II, LC3I, and LC3II/LC3I expressions determined by western blot. * P < .05, ** P < .001, vs Hep3B, # P < .05, ## P < .001, vs Hep3B/So, ^ P < .05, ^^ P < .001, vs Hep3B + So, △ P < .05, △△ P < .001, vs Hep3B/So + So, ▲ P < .05, ▲▲ P < .001, vs Hep3B/So + si‐SNHG16 + So, ‡ P < .05, ‡‡ P < .001, vs Hep3B/So + si‐SNHG16 + inhibitor + So

Article Snippet: 40 μg protein samples were separated on 10% SDS‐PAGE and then electrotransferred onto PVDF membrane, which was blocked by 5% skimmed milk at room temperature for 1 hour Next, the membrane was incubated with the primary antibodies LC3I (GTX17380, 1:1000; GeneTex) and LC3II (GTX127375, 1:3000; GeneTex), EGR1 (ab133695, 1:1000; Abcam) at 4°C overnight.

Techniques: Injection, Expressing, Quantitative RT-PCR, Western Blot