Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) Total cell lysates from MRC5 and other indicated NSCLC cell lines were immunoblotted with anti-EGR1. β-actin was used as loading control. (B) Representative of H1299 cells with or without overexpressed EGR1 immunofluorescence-stained with antibodies against EGR1 (red color). The nuclei were counterstained with DAPI (blue color). (C) The growth of H1299 cells infected with CD513B-1-EGR1 or -dnEGR1 was analyzed by CCK8 assay. Up panel: western blot for EGR1 expression levels in H1299 cells transfected with EGR1 or dnEGR1. (D) Histogram showing the percentages of apoptotic cells by flow cytometric staining with Annexin V-PE/7-AAD. (E) Western blot for apoptotic caspase-3 and -7 in H1299 cells with increasing quantities of EGR1 compared with control. Numbers presented the fold change compared with the vector. Relative values ± SD were obtained from three independent assays. The full-length blots of Fig. 1A, 1C and 1E were presented in .

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Immunofluorescence, Staining, Infection, CCK-8 Assay, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) The effect of EGR1 on cell migration was determined by wound healing assay. The spreading speed of EGR1-transfected cells along the wound edge was slower than that of vector- or dnEGR1-transfected cells over a period of 72 h. (C) Representative images showing the transfected cells that migrated through the cell. The number of migrated tumor cells was quantified and is shown in the right panel. Columns, mean of triplicate experiments; *P < 0.05.

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Migration, Wound Healing Assay, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A and B) Hierarchical clustering of 100 genes that exhibited significantly altered expression in EGR1-transfected H1299 cells as compared with vector-transfected H1299 cells. The color bar indicates the fold change (log 2 ). (C) IPA launch of bio-function enrichment profiles, plotted by relative statistical significance. P value, Fisher's exact test. (D) The expression of 15 upregulated genes (ARC, GDF15, CDKN1C, TGM2, HES1, EFNA1, NR4A1, TRIB1, IGFBP6, ITGB8, PDGFA, KRT18, TIMP1, and ID2) was analyzed by qPCR. Relative values ± SD were obtained from three independent assays.

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: Genes differentially expressed between EGR1-overexpressed H1299 cells and control cells

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Binding Assay, Dominant Negative Mutation, Expressing

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) Luciferase reporter assays of sequential deletions of the KRT18 promoter, cotransfected with increasing quantities of EGR1. The predicted positions of the EBS (black boxes) were indicated on the schematic. Nucleotides with red colors presented consensus EBS. Values are expressed as mean fold-activation to the promoter cotransfected with empty vector. (B) Luciferase reporter assays of point mutant of the KRT18 promoter. The consensus EBS in pGL3-KRT18 was mutated, creating pGL3-KRT18-mut. Relative reporter activity in response to EGR1 transfection was compared as in (A). (C) EGR1 and KRT18 protein levels after treatment with different si-EGR1 doses or NC (negative control). β-actin was used as loading control. Values are presented as fold change relative to NC. The full-length blot of Fig. 4C is presented in .

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Luciferase, Activation Assay, Plasmid Preparation, Mutagenesis, Activity Assay, Transfection, Negative Control

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) The growth of H1299 and A549 cells infected with CD513B-1-KRT18 was analyzed by CCK-8 assay. The results are expressed as mean ± SD of three independent experiments. (B) The effect of EGR1 on cell migration was determined by wound healing assay. (C) Representative images showing the transfected cells that migrated through the cell. The number of migrated tumor cells was quantified as in . (D) Western blot for apoptotic caspase-3 and -7 in H1299 cells with different KRT18 transfected dose as compared with control cells. β-actin was used as loading control. The full-length blot of Figure 5D is presented in . (E) Expression level of EGR1 and KRT18 in 2 primary NSCLC cases. (F) Correlation of the EGR1 (+) expression with age, lymph node metastasis and KRT18 in 36 NSCLC cases. (G) Correlation of the KRT18 (+) expression with lymph node metastasis in 36 NSCLC cases.

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Infection, CCK-8 Assay, Migration, Wound Healing Assay, Transfection, Western Blot, 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: 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: Oncology Reports

Article Title: Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis

doi: 10.3892/or.2023.8579

Figure Lengend Snippet: Effects of LNT on the EGR1/PTEN/AKT axis in Hepa1-6 cells. (A) Protein expression levels of EGR1, PTEN, p-Akt and phosphorylation of Akt after treatment with a gradient of LNT concentrations as detected by WB. All data for protein expression were normalized using β-actin as a loading reference. (B) Immunofluorescence co-staining was used to assess the localization and expression of EGR1 and PTEN in Hepa1-6 cells treated with a gradient of LNT concentrations. Images were observed at ×630 magnification. Scale bars, 50 µm (white). (C) WB was used to detect the expression of EGR1 in the nuclear and cytoplasmic fractions following treatment with a gradient of LNT concentrations. (D) WB was used to detect the expression of EGR1, PTEN, p-Akt, and phosphorylation of Akt after EGR1 overexpression. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. LNT, Lentinan; EGR1, early growth response 1; p-, phosphorylated; PARP1, poly-(ADP ribose) polymerase 1; Hsp60, heat shock protein 60; OE, overexpression; WB, western blotting.

Article Snippet: Subsequently, they were incubated with a mixture of different primary antibodies, including mouse EGR1 (1:100; cat. no. H00001958-M03; Novus Biologicals, LLC) and rabbit PTEN (1:100; cat. no. AF6351; Affinity Biosciences), overnight at 4°C.

Techniques: Expressing, Phospho-proteomics, Immunofluorescence, Staining, Over Expression, Western Blot

Journal: Oncology Reports

Article Title: Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis

doi: 10.3892/or.2023.8579

Figure Lengend Snippet: Inhibitory effect of LNT on DEN-induced primary liver cancer in mice. (A) The expression levels of EGR1, PTEN and other proteins in the liver tissues of the normal group and the model group, as analyzed by WB. (B) Expression of EGR1, PTEN and other proteins in liver tissues of the model and LNT-treated groups, as analyzed by WB. (C) Immunohistochemical analysis of EGR1, PTEN and Ki-67 in the tissue sections. Magnification, ×400. Scale bars, 200 µm (black). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. LNT, Lentinan; DEN, diethylnitrosamine; EGR1, early growth response 1; WB, western blotting; p-, phosphorylated; PCNA, proliferating cell nuclear antigen.

Article Snippet: Subsequently, they were incubated with a mixture of different primary antibodies, including mouse EGR1 (1:100; cat. no. H00001958-M03; Novus Biologicals, LLC) and rabbit PTEN (1:100; cat. no. AF6351; Affinity Biosciences), overnight at 4°C.

Techniques: Expressing, Immunohistochemical staining, Western Blot

Journal: Oncology Reports

Article Title: Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis

doi: 10.3892/or.2023.8579

Figure Lengend Snippet: Possible mechanism of LNT against liver cancer through the EGR1/PTEN/AKT axis. Blunt arrows indicate that the signaling pathways are inhibited, whilst pointed arrows indicate that the signaling pathways are activated. LNT, Lentinan; EGR1, early growth response 1.

Article Snippet: Subsequently, they were incubated with a mixture of different primary antibodies, including mouse EGR1 (1:100; cat. no. H00001958-M03; Novus Biologicals, LLC) and rabbit PTEN (1:100; cat. no. AF6351; Affinity Biosciences), overnight at 4°C.

Techniques: Protein-Protein interactions