Journal: The Journal of Biological Chemistry

Article Title: Egr1 mediates the effect of insulin on leptin transcription in adipocytes

doi: 10.1074/jbc.AC119.007855

Figure Lengend Snippet: Induction of Egr1 precedes leptin expression both in vitro and in vivo. A (top), 3T3-L1 adipocytes were treated with insulin (100 nm) and harvested at the indicated time points. Leptin and Egr1 mRNA levels were measured by qPCR. Bottom, Egr1 protein levels were measured in whole-cell lysates by Western blotting (50 μg/lane). B (top), male mice received an intraperitoneal injection of insulin (2 units/kg). Mice were sacrificed at the indicated time points, and total RNA was extracted from epididymal fat pads. Leptin mRNA levels were measured by qPCR. Bottom, Egr1 was detected in protein lysates of epididymal fat pads (50 μg/lane). C, following insulin injection (2 units/kg), mice were sacrificed, serum was collected by cardiac puncture, and circulating leptin was measured by ELISA. Data are presented as mean values ± S.D. (error bars). All panels show representative results of three independent experiments.

Article Snippet: Polyclonal antibody against Egr1 for ChIP was purchased from Novus Biologicals (Centennial, CO) (NBP1-78775).

Techniques: Expressing, In Vitro, In Vivo, Western Blot, Injection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Egr1 mediates the effect of insulin on leptin transcription in adipocytes

doi: 10.1074/jbc.AC119.007855

Figure Lengend Snippet: Egr1 activates leptin transcription in 3T3-L1 adipocytes in response to insulin stimulation. A, HEK 293T cells were transiently transfected with the pGL4.1 vector containing the leptin promoter linked to luciferase and increasing amounts of pcDNA-hEgr1 vector. B, 3T3-L1 adipocytes were treated with or without insulin for 4 h, and binding of Egr1 to the leptin promoter was analyzed by ChIP. C, HEK 293T cells were transiently transfected with pGL4.1 vector containing the leptin promoter linked to luciferase and either 500 ng of pcDNA-hEgr1 vector or a combination of 500 ng of pcDNA-hEgr1 and 500 ng of HA-FSP27 cDNA. D (top), 3T3-L1 adipocytes in suspension were transfected with SMARTpool Egr1 siRNA or scrambled siRNA at a final concentration of 100 nm for 72 h. Cells were then treated or not treated with insulin, and leptin mRNA was measured by qPCR. D (bottom), Egr1 was analyzed in whole-cell lysates by Western blotting (50 μg/lane). E (top), 3T3-L1 adipocytes were infected with adenovirus containing Egr1 (AdEgr1) and GFP (AdGFP) and cultured for 72 h. RNA was extracted, and leptin mRNA levels were analyzed by qPCR. E (bottom), Egr1 protein levels were measured in whole-cell lysates by Western blotting (50 μg/lane). Data are presented as mean values ± S.D. (error bars). All panels show representative results of three independent experiments: *, p < 0.05; **, p < 0.01; ***, p < 0.001, unpaired two-tailed t test.

Article Snippet: Polyclonal antibody against Egr1 for ChIP was purchased from Novus Biologicals (Centennial, CO) (NBP1-78775).

Techniques: Transfection, Plasmid Preparation, Luciferase, Binding Assay, Suspension, Concentration Assay, Western Blot, Infection, Cell Culture, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Egr1 mediates the effect of insulin on leptin transcription in adipocytes

doi: 10.1074/jbc.AC119.007855

Figure Lengend Snippet: Insulin activates leptin transcription in 3T3-L1 adipocytes primarily via the mTORC1-Egr1 axis. A (top), differentiated adipocytes were pretreated with PD98059 (15 μm) or PP242 (10 μm) for 30 min, and insulin (100 nm) was added for 4 h as indicated. Expression of the leptin mRNA was analyzed by qPCR. A (bottom), Egr1 protein levels were measured in whole-cell lysates by Western blotting (50 μg/lane). B, genomic DNA was extracted from WT 3T3-L1 cells and 3T3-L1 cells with deleted 5′-UTR of the Egr1 mRNA (Δ5′UTR) and amplified by PCR using 5′-UTR Egr1 primers. The dotted line indicates that irrelevant lanes have been spliced out. C, Egr1 mRNA levels were analyzed in WT 3T3-L1 adipocytes and Δ5′UTR 3T3-L1 adipocytes by qPCR. Data are expressed as the mean value normalized by 36B4 ± S.D. (error bars). D, WT 3T3-L1 adipocytes and Δ5′UTR 3T3-L1 adipocytes were treated with insulin (100 nm), and Egr1 was measured in whole-cell lysates by Western blotting (50 μg/lane). E, WT 3T3-L1 cells and Δ5′UTR 3T3-L1 cells were transfected with the pGL4.1 vector containing the leptin promoter linked to luciferase together with the pRL-CMV-Renilla vector on day 4 of differentiation, incubated for 72 h, and analyzed in a microplate reader. All panels show representative results of three independent experiments. Data are presented as mean values ± S.D.: *, p < 0.05; **, p < 0.01; ns, not significant, unpaired two-tailed t test.

Article Snippet: Polyclonal antibody against Egr1 for ChIP was purchased from Novus Biologicals (Centennial, CO) (NBP1-78775).

Techniques: Expressing, Western Blot, Amplification, Transfection, Plasmid Preparation, Luciferase, Incubation, Two Tailed Test

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

Journal: Frontiers in cell and developmental biology

Article Title: C9orf72 -Derived Proline:Arginine Poly-Dipeptides Modulate Cytoskeleton and Mechanical Stress Response.

doi: 10.3389/fcell.2022.750829

Figure Lengend Snippet: FIGURE 6 | PR20 prohibits the cyclic stretch-induced reorientation of F-actin stress fibers. (A) Western blotting shows Thbs1, Egr1, pFAK, FAK, pERK and ERK levels in CTRL and PR20-treated (PR) cells (n = 3). GAPDH was used as a loading control. (B) Quantification graphs are shown. Bars are means ± SEM. p value shows unpaired t-test. (C,E) Rat vascular SMCs (in C) and U2OS cells (in E) with or without 10 µM of PR20 were subjected to cyclic stretch (20% strain, 1.0 Hz (60 cycles/min) for 6 hours. Phalloidin (red) and DAPI (blue) are shown. Scale bars are 100 µm. The two-way arrows indicate stretch direction. (D,F) Histograms of the percentage of the orientation angle (θ) for each cell in rat vascular SMCs (in D) and U2OS cells (in F). The orientation of each cell was analyzed by measuring the orientation angle (θ) of the long axis of the ellipse relative to the stretch axis in CTRL (blue) and PR cells (red). 70 to 104 cells were evaluated in each condition. p value shows Mann–Whitney U test.

Article Snippet: Membranes were blocked with and incubated overnight at 4°Cwith the following primary antibodies: Thbs1 (NeoMarkers,MS421), Egr1 (Cell Signaling, 4154), phospho-FAK(Y397) (Cell Signaling, 3283), FAK (Cell Signaling, 3285), phospho-ERK (Cell Signaling, 4376), ERK (Cell Signaling, 9102), phospho-ERM (Cell Signaling, 3141), ERM (Cell Signaling, 3142), GAPDH (Cell Signaling, 2118).

Techniques: Western Blot, Control, MANN-WHITNEY

Journal: Genome Medicine

Article Title: Post-mortem molecular profiling of three psychiatric disorders

doi: 10.1186/s13073-017-0458-5

Figure Lengend Snippet: a Box plots indicating relative expression of EGR1 in the AnCg of SZ ( red ), BPD ( blue ), MDD ( green ), and CTL ( gray ). b Correlation plot comparing RNA-seq measured expression level of EGR1 to qPCR measured expression in ten SZ ( red ) and ten CTL ( black ) patients. c Wilcox p values resulting from comparing the degree of differential expression (based on DESeq2 p values) of genes whose TSS are within the indicated distance to an EGR1 binding sites compared to genes whose TSSs are further than the indicated threshold

Article Snippet: Validated Taqman assays for EGR1 (Hs00152928_m1) and the housekeeper genes GAPDH (Hs02758991_g1) and ACTB (Hs01060665_g1) were used for qPCR. cDNA was diluted by a factor of 10 before use as input for the Taqman assay.

Techniques: Expressing, RNA Sequencing, Quantitative Proteomics, Binding Assay