growth response protein 1 egr1  (Cell Signaling Technology Inc)

Journal: Marine drugs

Article Title: Chitotriose Enhanced Antitumor Activity of Doxorubicin through Egr1 Upregulation in MDA-MB-231 Cells.

doi: 10.3390/md22010026

Figure Lengend Snippet: Figure 6. Target genes and the key protein identified from composite treatment (n = 3 in each group). (A) Venn diagram analysis indicated the number of genes of overlapping groups. In total, 411 common genes were further used for PPI analysis. (B) PPI analysis indicated EGR1 as the key regulatory protein.

Article Snippet: The antibodies for early growth response protein 1 (EGR1) (catalogue number: 4154S), growth arrest, and the DNA damage-inducible protein alpha (GADD45A) (catalogue number: 4632S), and β-actin (catalogue number: 4970S) were procured from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques:

Journal: Marine drugs

Article Title: Chitotriose Enhanced Antitumor Activity of Doxorubicin through Egr1 Upregulation in MDA-MB-231 Cells.

doi: 10.3390/md22010026

Figure Lengend Snippet: Figure 7. The effect of chitotriose on the transcription and expression levels of Egr1 and Gadd45a (n = 3 in each group) with siRNA transfection. The mRNA level of (A) Egr1 and (B) Gadd45a detected via RT-qPCR. Asterisks (***) indicate significant differences at p-value < 0.001 compared with the CTL group. Sharps (#), (##), and (###) indicate significant differences at p-values < 0.05, 0.01 and 0.001 for each group before and after siRNA transfection. (C) Protein levels detected using the Western blot assay. (D) Cell viability detected via the CCK-8 assay. Sharps (###) indicate significant differences at p-value < 0.001 between the combined and DOX groups.

Article Snippet: The antibodies for early growth response protein 1 (EGR1) (catalogue number: 4154S), growth arrest, and the DNA damage-inducible protein alpha (GADD45A) (catalogue number: 4632S), and β-actin (catalogue number: 4970S) were procured from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: GPR43 activation-mediated lipotoxicity contributes to podocyte injury in diabetic nephropathy by modulating the ERK/EGR1 pathway

doi: 10.7150/ijbs.64665

Figure Lengend Snippet: GPR43 activation inhibited LDLR-mediated cholesterol uptake and autophagy-mediated cholesterol degradation in podocytes in DN by modulating the EGR1 pathway. A. The protein expression of EGR1 in kidneys was examined by Western blotting. The mice were divided into four groups: the control group (Ctrl, n=6), GPR43-KO group (n=6), diabetes group (DM, n=6), and GPR43-KO diabetes mice (GPR43-KO+DM, n=7). The mice were treated for 12 weeks. The histogram shows the means ± SD of the densitometric scans of EGR1 protein bands following normalization to β-actin. * P < 0.05 vs . Ctrl, # P < 0.05 vs . DM. B. The protein expression of EGR1 in podocytes in kidneys was examined by immunofluorescent staining. The mice were divided into four groups: the control group (Ctrl, n=6), GPR43-KO group (n=6), diabetes group (DM, n=6), and GPR43-KO diabetes mice (GPR43-KO+DM, n=7). The mice were treated for 12 weeks. The fluorescent signals of EGR1 (green), WT-1 (red), and nuclei (blue) were examined by confocal microscopy (original magnification × 400, scale bars, 50 µm). EGR1: early growth response protein 1. C. The protein expression of EGR1 in podocytes was examined by Western blotting. Podocytes were treated with different concentrations of acetate in the presence of high glucose (HG, 30 mmol/L) for 24 hours. The histogram shows the means ± SD of the densitometric scans of EGR1 protein bands following normalization to β-actin. * P < 0.05 vs. HG. D. The protein expression of EGR1 in podocytes was examined by Western blotting. Podocytes were treated with or without acetate, GPR43 siRNA, or control siRNA (vehicle) for 24 hours in the presence of high glucose conditions (HG, 30 mmol/L). The histogram shows the means ± SD of the densitometric scans of EGR1 protein bands following normalization to β-actin. * P < 0.05 vs . HG + control siRNA; ## P < 0.01 vs . HG + acetate + control siRNA. E. The mRNA and protein expression of EGR1 in podocytes was respectively examined by real-time PCR and Western blotting. Podocytes were treated with or without acetate, EGR1 siRNA, or control siRNA (vehicle) for 24 hours in the presence of high glucose conditions (HG, 30 mmol/L). The histogram shows the means ± SD of the densitometric scans of EGR1 protein bands following normalization to β-actin. ** P < 0.01 vs. HG + control siRNA; ## P < 0.01 vs. HG + acetate + control siRNA. F. Cholesterol accumulation in podocytes was examined by quantitative free cholesterol assays. Podocytes were treated with or without acetate, EGR1 siRNA, or control siRNA (vehicle) for 24 hours in the presence of high glucose conditions (HG, 30 mmol/L). The concentration of free cholesterol was normalized to the total protein in cells (mean ± SD, * P < 0.05 vs. HG + control siRNA, # P < 0.05 vs. HG + acetate + control siRNA). G-H. The protein expression of LC3 (G) and LDLR (H) in podocytes was examined by Western blotting. Podocytes were treated with or without acetate, EGR1 siRNA, or control siRNA (vehicle) for 24 hours in the presence of high glucose conditions (HG, 30 mmol/L). The histogram shows the means ± SD of the densitometric scans of LC3 and LDLR protein bands following normalization to β-actin. * P < 0.05 vs. HG + control siRNA; # P < 0.05 vs. HG + acetate + control siRNA.

Article Snippet: The summarized primary antibodies were used: GPR43 (Sigma-Aldrich), WT-1(Santa Cruz), extracellular regulated kinase (ERK) 1/2 and phosphorylated ERK1/2 (Cell Signaling Technology), early growth response protein 1 (EGR1) and LC3 (Proteintech), p62 and LDLR (Abcam), and secondary antibodies against goat anti-rabbit IgG (H+L) (Alexa Fluor 488) and goat anti-mouse IgG (H+L) (Alexa Fluor 594) (Proteintech) were used.

Techniques: Activation Assay, Expressing, Western Blot, Control, Staining, Confocal Microscopy, Real-time Polymerase Chain Reaction, Concentration Assay

Journal: International Journal of Biological Sciences

Article Title: GPR43 activation-mediated lipotoxicity contributes to podocyte injury in diabetic nephropathy by modulating the ERK/EGR1 pathway

doi: 10.7150/ijbs.64665

Figure Lengend Snippet: GPR43 activation upregulated the expression and transcriptional activity of EGR1 in podocytes of DN by increasing the nuclear translocation of ERK1/2. A. ERK1/2 protein phosphorylation in kidneys was examined by Western blotting. The mice were divided into four groups: the control group (Ctrl, n=6), GPR43-KO group (n=6), diabetes group (DM, n=6), and GPR43-KO diabetes mice (GPR43-KO+DM, n=7). The mice were treated for 12 weeks. The histogram shows the means ± SD of the densitometric scans of phosphorylated ERK1/2 protein bands following normalization to β-actin ( ** P < 0.01 vs . Ctrl, ## P < 0.01 vs . DM). B. ERK1/2 protein phosphorylation in podocytes was examined by Western blotting. Podocytes were treated with different concentrations of acetate in the presence of high glucose (HG, 30 mmol/L) for 24 hours. The histogram shows the means ± SD of the densitometric scans of ERK1/2 phosphorylated protein bands following normalization to β-actin. * P < 0.05, ** P < 0.01 vs . HG. C. ERK1/2 protein phosphorylation in podocytes was examined by Western blotting. Podocytes were treated with or without acetate, GPR43 siRNA, or control siRNA (vehicle) for 24 hours in the presence of high glucose conditions (HG, 30 mmol/L). The histogram shows the means ± SD of the densitometric scans of ERK1/2 phosphorylated protein bands following normalization to β-actin. * P < 0.05 vs. HG + control siRNA; # P < 0.05 vs. HG + acetate + control siRNA. D. Cholesterol accumulation in podocytes was examined by quantitative free cholesterol assays. Podocytes were treated with or without acetate and a MEK inhibitor (PD0325901, 5 µmol/L) for 24 hours in the presence of high glucose conditions (HG, 30 mmol/L). The concentration of free cholesterol was normalized to the total protein in cells. (mean ± SD, * P < 0.05 vs. HG; ## P <0.01 vs. HG + acetate). E. The protein expression of pERK1/2, EGR1, LDLR, and LC3 in podocytes was examined by Western blotting. Podocytes were treated with or without acetate and a MEK inhibitor (PD0325901, 5 µmol/L) for 24 hours in the presence of high glucose conditions (HG, 30 mmol/L). The histogram shows the means ± SD of the densitometric scans of the pERK1/2, EGR1, LDLR and LC3 protein bands following normalization to β-actin. * P < 0.05 vs. HG; # P < 0.05 vs. HG + acetate.

Article Snippet: The summarized primary antibodies were used: GPR43 (Sigma-Aldrich), WT-1(Santa Cruz), extracellular regulated kinase (ERK) 1/2 and phosphorylated ERK1/2 (Cell Signaling Technology), early growth response protein 1 (EGR1) and LC3 (Proteintech), p62 and LDLR (Abcam), and secondary antibodies against goat anti-rabbit IgG (H+L) (Alexa Fluor 488) and goat anti-mouse IgG (H+L) (Alexa Fluor 594) (Proteintech) were used.

Techniques: Activation Assay, Expressing, Activity Assay, Translocation Assay, Phospho-proteomics, Western Blot, Control, Concentration Assay

Journal: International Journal of Biological Sciences

Article Title: GPR43 activation-mediated lipotoxicity contributes to podocyte injury in diabetic nephropathy by modulating the ERK/EGR1 pathway

doi: 10.7150/ijbs.64665

Figure Lengend Snippet: Schematic diagram showing that GPR43-mediated lipotoxicity contributed to podocyte injury in DN. Under high glucose conditions, acetate-mediated GPR43 activation stimulated cholesterol uptake in podocytes through the upregulation of LDLR and inhibited cholesterol hydrolysis and degradation by inhibiting autophagy. The potential mechanism was mainly mediated by modulation of the GPR43/ERK/EGR1 pathway. Moreover, EGR1 activation was a critical factor that governs both LDLR-mediated cholesterol uptake and autophagy-mediated cholesterol degradation, ultimately leading to cholesterol accumulation in podocytes and DN progression.

Article Snippet: The summarized primary antibodies were used: GPR43 (Sigma-Aldrich), WT-1(Santa Cruz), extracellular regulated kinase (ERK) 1/2 and phosphorylated ERK1/2 (Cell Signaling Technology), early growth response protein 1 (EGR1) and LC3 (Proteintech), p62 and LDLR (Abcam), and secondary antibodies against goat anti-rabbit IgG (H+L) (Alexa Fluor 488) and goat anti-mouse IgG (H+L) (Alexa Fluor 594) (Proteintech) were used.

Techniques: Activation Assay

Journal: International journal of oncology

Article Title: Identification of phenothiazine as an ETV1‑targeting agent in gastrointestinal stromal tumors using the Connectivity Map.

doi: 10.3892/ijo.2019.4829

Figure Lengend Snippet: Figure 7. Western blot analysis of (A) GIST882 and (B) GIST48 cells following treatment with either TFP or TDZ, or in combination with MEK162. Increased LC3‑II expression was found following TFP and TDZ treatment with concomitant activation of ERK, ELK1 and EGR1. Combination of MEK162 and pheno thiazine diminished autophagy, enhanced apoptosis, and downregulate ERK and downstream ELK1 and EGR1.

Article Snippet: The primary antibodies used for western blot analysis included p‐ERK (cat. no. 4376; 1:1,000), KIT (cat. no. 3392; 1:1,000), p‐KIT (cat. no. 3391; 1:1,000), AKT (cat. no. 9272; 1:1,000), p‐AKT (cat. no. 4058; 1:1,000), p‐p70S6K (cat. no. 9205; 1:500), LC3 (cat. no. 2775; 1:1,000), early growth response protein 1 (EGR1) (cat. no. 4153; 1:1,000) and poly-(ADP-ribose) polymerase (PARP) (cat. no. 9542; 1:1,000) from Cell Signaling Technology; actin (cat. no. ab6276‐100; 1:10,000) and ETV1 (cat. no. ab184120; 1:1,000) from Abcam Biotechnology; and ERK (cat. no. sc-135900; 1:1,000), ELK1 (cat. no. sc-355; 1:200) and p-ELK1 (cat. no. sc-8406; 1:200) from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Activation Assay