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Proteintech rabbit polyclonal anti egr1
Rabbit Polyclonal Anti Egr1, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+egr1/pm41764198-251-39-44?v=Proteintech
Average 96 stars, based on 114 article reviews

rabbit polyclonal anti egr1 - by Bioz Stars, 2026-08

96/100 stars

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EIF2AK2 mediates LDLR expression in the ribosome-insulted gut. (A-D) Small intestine-derived organoids from the wild-type mice were treated with vehicle, RIS-1 (500 ng/mL), RIS-2 (1 μM), or EIF2AK2 inhibitor (0.5 μM, EIF2AK2i) for 24 h. (A) Representative Oil Red O staining for the intestinal organoids. The microscopy analysis was performed at the original magnification of 100×; scale bar(s), 100 μm. (B) The graph shows relative quantitative values of Oil Red O-positive lipid droplets in the gut organoids using ImageJ software. Different letters over each bar represent significant differences between groups ( p < 0.05). (C) The confocal microscopic analysis of LDLR expression was performed at the original magnification of 200×; scale bar(s), 50 μm. (D) The graph shows the relative quantitative values for LDLR protein in the intestinal organoids using ImageJ software. Different letters over each bar represent significant differences between groups ( p < 0.05). (E-G) HCT-8 cells transfected with the negative control vector or shEIF2AK2 were treated with the vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2. (E) Intracellular lipid droplets were stained with Oil Red O and visualized using a light microscope. The microscopy analysis was performed at the original magnification of 200×; scale bar(s), 50 μm. (F) The graph shows relative quantitative values of Oil Red O-positive lipid droplets in cells using ImageJ software (*** p < 0.001). (G) HCT-8 cells expressing the negative control or EGR1-specific shRNA were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. LDLR mRNA was measured using reverse transcription-quantitative PCR (*** p < 0.001).

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: EIF2AK2 mediates LDLR expression in the ribosome-insulted gut. (A-D) Small intestine-derived organoids from the wild-type mice were treated with vehicle, RIS-1 (500 ng/mL), RIS-2 (1 μM), or EIF2AK2 inhibitor (0.5 μM, EIF2AK2i) for 24 h. (A) Representative Oil Red O staining for the intestinal organoids. The microscopy analysis was performed at the original magnification of 100×; scale bar(s), 100 μm. (B) The graph shows relative quantitative values of Oil Red O-positive lipid droplets in the gut organoids using ImageJ software. Different letters over each bar represent significant differences between groups ( p < 0.05). (C) The confocal microscopic analysis of LDLR expression was performed at the original magnification of 200×; scale bar(s), 50 μm. (D) The graph shows the relative quantitative values for LDLR protein in the intestinal organoids using ImageJ software. Different letters over each bar represent significant differences between groups ( p < 0.05). (E-G) HCT-8 cells transfected with the negative control vector or shEIF2AK2 were treated with the vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2. (E) Intracellular lipid droplets were stained with Oil Red O and visualized using a light microscope. The microscopy analysis was performed at the original magnification of 200×; scale bar(s), 50 μm. (F) The graph shows relative quantitative values of Oil Red O-positive lipid droplets in cells using ImageJ software (*** p < 0.001). (G) HCT-8 cells expressing the negative control or EGR1-specific shRNA were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. LDLR mRNA was measured using reverse transcription-quantitative PCR (*** p < 0.001).

Article Snippet: The membranes were then incubated with the following antibodies: rabbit polyclonal anti-human actin antibody (1:1000), rabbit polyclonal anti-human SREPB2 antibody (1:1000), rabbit polyclonal anti-human EGR1 antibody (1:1000), mouse monoclonal anti-human HuR antibody (1:2000), mouse monoclonal anti-human hnRNP antibody (1:2000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-human LDLR antibody (1:1000) (Cayman Chemical, Ann Arbor, Michigan, USA) for 2 h at room temperature.

Techniques: Expressing, Derivative Assay, Staining, Microscopy, Software, Transfection, Negative Control, Plasmid Preparation, Light Microscopy, shRNA, Reverse Transcription, Real-time Polymerase Chain Reaction

$LDLR expression in response to ribosomal inactivation is dependent on EGR1 and SREBP2. (A) HCT-8 cells were treated with 1000 ng/mL RIS-1 for the indicated time. The graph shows the quantification of nuclear SREBP2 protein from the confocal microscopic analysis (boxed panels) and LDLR mRNA using reverse transcription-quantitative PCR (RT-qPCR). (B) HCT-8 cells were pretreated with the vehicle, 10 μM SP600125 (SP), 10 μM SB203580 (SB), or 2 μM U0126 for 2 h and then treated with vehicle or 1000 ng/mL RIS-1 for 30 min. The bar graph shows the quantification of nuclear SREBP2 protein using a confocal microscope (** p < 0.01 and *** p < 0.001). (C and D) For the ChIP assay, HCT-8 cells were treated with vehicle or 1000 ng/mL RIS-1 for the indicated time. (E) HCT-8 cells were treated with vehicle or 1000 ng/mL RIS-1 for the indicated time and examined under a confocal microscope. The microscopic analysis was performed at the original magnification of 1600×; scale bar(s), 20 μm. The right panel shows the relative quantitative values for nuclear EGR1 protein in cells. The relative ratio was obtained by measuring the nuclear density of EGR1 corresponding to the DAPI-stained area (the right graph, * p < 0.05). (F) HCT-8 cells were treated with vehicle or 1000 ng/mL RIS-1 for the indicated time. Nuclear fractions of cell lysates were subjected to western blot analysis. (G) HCT-8 cells pretreated with the vehicle, 10 μM SP600125 (SP), 10 μM SB203580 (SB), or 2 μM U0126 for 2 h were exposed to vehicle or 1000 ng/mL RIS-1 for 1 h. Nuclear fractions of cell lysates were subjected to western blot analysis. ChIP, chromatin immunoprecipitation; EGR1, early growth response 1; LDLR, low-density lipoprotein receptor; SREBP2, sterol regulatory element-binding protein 2.$

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: LDLR expression in response to ribosomal inactivation is dependent on EGR1 and SREBP2. (A) HCT-8 cells were treated with 1000 ng/mL RIS-1 for the indicated time. The graph shows the quantification of nuclear SREBP2 protein from the confocal microscopic analysis (boxed panels) and LDLR mRNA using reverse transcription-quantitative PCR (RT-qPCR). (B) HCT-8 cells were pretreated with the vehicle, 10 μM SP600125 (SP), 10 μM SB203580 (SB), or 2 μM U0126 for 2 h and then treated with vehicle or 1000 ng/mL RIS-1 for 30 min. The bar graph shows the quantification of nuclear SREBP2 protein using a confocal microscope (** p < 0.01 and *** p < 0.001). (C and D) For the ChIP assay, HCT-8 cells were treated with vehicle or 1000 ng/mL RIS-1 for the indicated time. (E) HCT-8 cells were treated with vehicle or 1000 ng/mL RIS-1 for the indicated time and examined under a confocal microscope. The microscopic analysis was performed at the original magnification of 1600×; scale bar(s), 20 μm. The right panel shows the relative quantitative values for nuclear EGR1 protein in cells. The relative ratio was obtained by measuring the nuclear density of EGR1 corresponding to the DAPI-stained area (the right graph, * p < 0.05). (F) HCT-8 cells were treated with vehicle or 1000 ng/mL RIS-1 for the indicated time. Nuclear fractions of cell lysates were subjected to western blot analysis. (G) HCT-8 cells pretreated with the vehicle, 10 μM SP600125 (SP), 10 μM SB203580 (SB), or 2 μM U0126 for 2 h were exposed to vehicle or 1000 ng/mL RIS-1 for 1 h. Nuclear fractions of cell lysates were subjected to western blot analysis. ChIP, chromatin immunoprecipitation; EGR1, early growth response 1; LDLR, low-density lipoprotein receptor; SREBP2, sterol regulatory element-binding protein 2.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Microscopy, Staining, Western Blot, Chromatin Immunoprecipitation, Binding Assay

Transcriptional regulation of LDLR expression in response to ribosomal inactivation. (A) A putative scheme for ribosomal stress-induced transcriptional activation of LDLR expression via SREBP2 activation and EGR1 induction. (B) HCT-8 cells expressing the negative control vector, EGR1-, or SREBP2-specific shRNA were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. LDLR mRNA was measured using reverse transcription-quantitative PCR. Figures in the box represent the inhibition of EGR-1 and SREBP2 mRNA by each shRNA (*** p < 0.001). (C) HCT-8 cells transfected with the wild-type (wt) or the mutants (SRE mt and CRE mt) of LDLR promoter-containing plasmids were treated with vehicle or 1000 ng/mL RIS-1 for 6 h, and the cellular luciferase activity was measured (* p < 0.05). (D) EGR1- or SREBP2-deficient cells were transiently transfected with wt LDLR promoter-containing plasmid and then treated with vehicle or 1000 ng/mL RIS-1 for 6 h to measure the cellular LDLR promoter activity. Different letters over each bar represent significant differences between the two groups ( p < 0.05). The right boxed graphs show suppression of mRNA expression by each shRNA (*** p < 0.001). (E-F) HCT-8 cells expressing the negative control vector, EGR1-, or SREBP2-specific shRNA were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. Intracellular lipid droplets were stained with Oil Red O and visualized using a light microscope at the original magnification of 200×; scale bar(s), 50 μm (E). The right graph shows the relative quantitative values of Oil Red O-positive lipid droplets, and different letters over each bar represent significant differences between the two groups (F, p < 0.05 using one-way ANOVA with the Newman-Keuls post hoc test). EGR1, early growth response 1; LDLR, low-density lipoprotein receptor; SREBP2, sterol regulatory element-binding protein 2.

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: Transcriptional regulation of LDLR expression in response to ribosomal inactivation. (A) A putative scheme for ribosomal stress-induced transcriptional activation of LDLR expression via SREBP2 activation and EGR1 induction. (B) HCT-8 cells expressing the negative control vector, EGR1-, or SREBP2-specific shRNA were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. LDLR mRNA was measured using reverse transcription-quantitative PCR. Figures in the box represent the inhibition of EGR-1 and SREBP2 mRNA by each shRNA (*** p < 0.001). (C) HCT-8 cells transfected with the wild-type (wt) or the mutants (SRE mt and CRE mt) of LDLR promoter-containing plasmids were treated with vehicle or 1000 ng/mL RIS-1 for 6 h, and the cellular luciferase activity was measured (* p < 0.05). (D) EGR1- or SREBP2-deficient cells were transiently transfected with wt LDLR promoter-containing plasmid and then treated with vehicle or 1000 ng/mL RIS-1 for 6 h to measure the cellular LDLR promoter activity. Different letters over each bar represent significant differences between the two groups ( p < 0.05). The right boxed graphs show suppression of mRNA expression by each shRNA (*** p < 0.001). (E-F) HCT-8 cells expressing the negative control vector, EGR1-, or SREBP2-specific shRNA were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. Intracellular lipid droplets were stained with Oil Red O and visualized using a light microscope at the original magnification of 200×; scale bar(s), 50 μm (E). The right graph shows the relative quantitative values of Oil Red O-positive lipid droplets, and different letters over each bar represent significant differences between the two groups (F, p < 0.05 using one-way ANOVA with the Newman-Keuls post hoc test). EGR1, early growth response 1; LDLR, low-density lipoprotein receptor; SREBP2, sterol regulatory element-binding protein 2.

Techniques: Expressing, Activation Assay, Negative Control, Plasmid Preparation, shRNA, Reverse Transcription, Real-time Polymerase Chain Reaction, Inhibition, Transfection, Luciferase, Activity Assay, Staining, Light Microscopy, Binding Assay

Effects of ribosomal inactivation on LDLR mRNA stability in human intestinal cells. (A) HCT-8 cells were treated with the vehicle or 1000 ng/mL RIS-1 for 2 h and then replaced with 5 μM actinomycin D for the indicated time to arrest cellular transcription. Expression levels of LDLR mRNA were measured using reverse transcription-quantitative PCR (RT-qPCR). The underlying boxed blots represent the mRNA measured by conventional RT-PCR. (B) HCT-8 cells were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h and examined using a confocal microscope. The microscopic analysis was performed at the original magnification of 1600×; scale bar(s), 20 μm. The right graph shows the relative quantitative values of cytosolic HuR in cells using ImageJ software (*** p < 0.001). (C) HCT-8 cells transfected with the negative control vector or HuR shRNA expression vector (shHuR) were treated with the vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. mRNA levels were measured using RT-qPCR. The boxed images show the suppression of HuR mRNA expression by shHuR (*** p < 0.001). (D) A putative scheme for ribosomal inactivation-induced cholesterol uptake. (E) Pearson 's correlation analysis of relationships between transcription levels of critical components of ribosotoxic stress responses and LDLR in the small intestine of diet-induced obese mice (gse199776). Correlation matrix visualization was generated using the corrplot function of the R-package (R Foundation for Statistical Computing, Vienna, Austria. URL: https://www.R-project.org/ ). Correlations of transcriptional expression among genes were interpreted according to a general guideline for Pearson's coefficient value: r > 0.7, high (+); 0.5 < r < 0.7, moderate (+); 0.3 < r < 0.5, moderate (+) or low (+); 0.1 < r < 0.3, low (+); -0.1 < r < 0.1, negligible; -0.3 < r < -0.1, low (-). (F) LDLR expression was assessed in patients with IBD (gse75214; Vemeire 's, n = 194). Based on EGR1, SREBP , or HuR levels, we selected samples exhibiting the 50 highest and 50 lowest levels, which were further compared for LDLR levels. Results are shown as a box-and-whisker plot (Tukey), and asterisks (*) indicate significant differences from the low expression group (* p < 0.05, *** p < 0.001). EGR1, early growth response 1; HuR, human antigen R; IBD, inflammatory bowel disease; LDL, low-density lipoprotein; LDL, low-density lipoprotein receptor; SREBP, sterol regulatory element-binding protein.

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: Effects of ribosomal inactivation on LDLR mRNA stability in human intestinal cells. (A) HCT-8 cells were treated with the vehicle or 1000 ng/mL RIS-1 for 2 h and then replaced with 5 μM actinomycin D for the indicated time to arrest cellular transcription. Expression levels of LDLR mRNA were measured using reverse transcription-quantitative PCR (RT-qPCR). The underlying boxed blots represent the mRNA measured by conventional RT-PCR. (B) HCT-8 cells were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h and examined using a confocal microscope. The microscopic analysis was performed at the original magnification of 1600×; scale bar(s), 20 μm. The right graph shows the relative quantitative values of cytosolic HuR in cells using ImageJ software (*** p < 0.001). (C) HCT-8 cells transfected with the negative control vector or HuR shRNA expression vector (shHuR) were treated with the vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. mRNA levels were measured using RT-qPCR. The boxed images show the suppression of HuR mRNA expression by shHuR (*** p < 0.001). (D) A putative scheme for ribosomal inactivation-induced cholesterol uptake. (E) Pearson 's correlation analysis of relationships between transcription levels of critical components of ribosotoxic stress responses and LDLR in the small intestine of diet-induced obese mice (gse199776). Correlation matrix visualization was generated using the corrplot function of the R-package (R Foundation for Statistical Computing, Vienna, Austria. URL: https://www.R-project.org/ ). Correlations of transcriptional expression among genes were interpreted according to a general guideline for Pearson's coefficient value: r > 0.7, high (+); 0.5 < r < 0.7, moderate (+); 0.3 < r < 0.5, moderate (+) or low (+); 0.1 < r < 0.3, low (+); -0.1 < r < 0.1, negligible; -0.3 < r < -0.1, low (-). (F) LDLR expression was assessed in patients with IBD (gse75214; Vemeire 's, n = 194). Based on EGR1, SREBP , or HuR levels, we selected samples exhibiting the 50 highest and 50 lowest levels, which were further compared for LDLR levels. Results are shown as a box-and-whisker plot (Tukey), and asterisks (*) indicate significant differences from the low expression group (* p < 0.05, *** p < 0.001). EGR1, early growth response 1; HuR, human antigen R; IBD, inflammatory bowel disease; LDL, low-density lipoprotein; LDL, low-density lipoprotein receptor; SREBP, sterol regulatory element-binding protein.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Microscopy, Software, Transfection, Negative Control, Plasmid Preparation, shRNA, Generated, Whisker Assay, Binding Assay

The forward- and reverse-complement PCR primers for amplification of each gene

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: The forward- and reverse-complement PCR primers for amplification of each gene

Techniques: Amplification, Sequencing

The gene sequences used for transfection.

Journal: International Journal of Endocrinology

Article Title: Metformin Regulating miR-34a Pathway to Inhibit Egr1 in Rat Mesangial Cells Cultured with High Glucose

doi: 10.1155/2018/6462793

Figure Lengend Snippet: The gene sequences used for transfection.

Article Snippet: The antibodies used in this study include rabbit polyclonal anti-FN (H-300), rabbit polyclonal anti-SIRT1 (H-300), rabbit polyclonal anti-Egr1 (C-19), rabbit polyclonal anti-CTGF (H-55), and rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (FL-335) from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and rabbit monoclonal anti-AMPK α (4188S) and rabbit monoclonal anti-phospho-AMPK α (Thr172) (4811S) from Cell Signaling Technology (USA).

Techniques: Transfection

Primer sequences used for real-time PCR primers.

Journal: International Journal of Endocrinology

Article Title: Metformin Regulating miR-34a Pathway to Inhibit Egr1 in Rat Mesangial Cells Cultured with High Glucose

doi: 10.1155/2018/6462793

Figure Lengend Snippet: Primer sequences used for real-time PCR primers.

Techniques: Real-time Polymerase Chain Reaction

The effects of high glucose on Egr1 mRNA (a) and protein (b) expression. RMCs were incubated with high glucose (30 mmol/L glucose) for 0–24 hours. mRNA expression was determined by quantitative real-time PCR, and protein expression was determined by western blotting. ∗ p < 0.05, ∗∗ p < 0.001 versus time zero control. All results represent means ± SD obtained from three independent experiments in triplicate. Egr1: early growth response factor 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Journal: International Journal of Endocrinology

Article Title: Metformin Regulating miR-34a Pathway to Inhibit Egr1 in Rat Mesangial Cells Cultured with High Glucose

doi: 10.1155/2018/6462793

Figure Lengend Snippet: The effects of high glucose on Egr1 mRNA (a) and protein (b) expression. RMCs were incubated with high glucose (30 mmol/L glucose) for 0–24 hours. mRNA expression was determined by quantitative real-time PCR, and protein expression was determined by western blotting. ∗ p < 0.05, ∗∗ p < 0.001 versus time zero control. All results represent means ± SD obtained from three independent experiments in triplicate. Egr1: early growth response factor 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Western Blot

High glucose obviously suppresses SIRT1 protein and the p-AMPK α /AMPK α ratio (by western blotting) in RMCs and increases Egr1 protein (by western blotting) in RMCs. RMCs were incubated with normal glucose (NG, 5.5 mmol/L glucose), high mannitol (HM, 5.5 mmol/L glucose + 24.5 mmol/L mannitol), or high glucose (HG, 30 mmol/L glucose). ∗ p < 0.001 versus NG control. All results represent means ± SD obtained from three independent experiments in triplicate and normalized to GAPDH. SIRT1: sirtuin 1; Egr1: early growth response factor 1; p-AMPK α : phosphorylated adenosine monophosphate-activated protein kinase α ; AMPK α : adenosine monophosphate-activated protein kinase α ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Journal: International Journal of Endocrinology

Article Title: Metformin Regulating miR-34a Pathway to Inhibit Egr1 in Rat Mesangial Cells Cultured with High Glucose

doi: 10.1155/2018/6462793

Figure Lengend Snippet: High glucose obviously suppresses SIRT1 protein and the p-AMPK α /AMPK α ratio (by western blotting) in RMCs and increases Egr1 protein (by western blotting) in RMCs. RMCs were incubated with normal glucose (NG, 5.5 mmol/L glucose), high mannitol (HM, 5.5 mmol/L glucose + 24.5 mmol/L mannitol), or high glucose (HG, 30 mmol/L glucose). ∗ p < 0.001 versus NG control. All results represent means ± SD obtained from three independent experiments in triplicate and normalized to GAPDH. SIRT1: sirtuin 1; Egr1: early growth response factor 1; p-AMPK α : phosphorylated adenosine monophosphate-activated protein kinase α ; AMPK α : adenosine monophosphate-activated protein kinase α ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Techniques: Western Blot, Incubation

Activating AMPK α significantly reduces Egr1 expression (by western blotting) stimulated by high glucose in RMCs. RMCs were pre-incubated with 1 mmol/L AMPK α activator AICAR (+AI) for 30 minutes before stimulating with normal glucose (NG, 5.5 mmol/L glucose) or high glucose (HG, 30 mmol/L glucose). ∗ p < 0.05, ∗∗ p < 0.001 versus RMCs cultured with normal glucose media. # p < 0.001 versus RMCs cultured with high glucose media. All results represent means ± SD obtained from three independent experiments in triplicate and normalized to GAPDH. AICAR: 5-amino-4-imidazolecarboxamide riboside-1-b-D-ribofuranoside; Egr1: early growth response factor 1; p-AMPK α : phosphorylated adenosine monophosphate-activated protein kinase α ; AMPK α : adenosine monophosphate-activated protein kinase α ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Journal: International Journal of Endocrinology

Article Title: Metformin Regulating miR-34a Pathway to Inhibit Egr1 in Rat Mesangial Cells Cultured with High Glucose

doi: 10.1155/2018/6462793

Figure Lengend Snippet: Activating AMPK α significantly reduces Egr1 expression (by western blotting) stimulated by high glucose in RMCs. RMCs were pre-incubated with 1 mmol/L AMPK α activator AICAR (+AI) for 30 minutes before stimulating with normal glucose (NG, 5.5 mmol/L glucose) or high glucose (HG, 30 mmol/L glucose). ∗ p < 0.05, ∗∗ p < 0.001 versus RMCs cultured with normal glucose media. # p < 0.001 versus RMCs cultured with high glucose media. All results represent means ± SD obtained from three independent experiments in triplicate and normalized to GAPDH. AICAR: 5-amino-4-imidazolecarboxamide riboside-1-b-D-ribofuranoside; Egr1: early growth response factor 1; p-AMPK α : phosphorylated adenosine monophosphate-activated protein kinase α ; AMPK α : adenosine monophosphate-activated protein kinase α ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Techniques: Expressing, Western Blot, Incubation, Cell Culture

miR-34a inhibitor significantly suppresses miR-34a expression (by quantitative real-time PCR) stimulated by high glucose (a). And miR-34a inhibitor activates SIRT1/AMPK α and restrains Egr1 expression (by western blotting) in high glucose conditions (b) in RMCs. RMCs were transfected with miR-34a inhibitor (IN) or nonspecific control (N-NC) for 6 hours and further cultured with normal glucose (NG, 5.5 mmol/L glucose) or high glucose (HG, 30 mmol/L glucose) media for 24 hours. ∗ p < 0.05, ∗∗ p < 0.001 versus RMCs cultured with normal glucose media. # p < 0.001 versus RMCs cultured with high glucose media. All results represent means ± SD obtained from three independent experiments in triplicate. And western blotting results were normalized to GAPDH. SIRT1: sirtuin 1; Egr1: early growth response factor 1; p-AMPK α : phosphorylated adenosine monophosphate-activated protein kinase α ; AMPK α : adenosine monophosphate-activated protein kinase α ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Journal: International Journal of Endocrinology

Article Title: Metformin Regulating miR-34a Pathway to Inhibit Egr1 in Rat Mesangial Cells Cultured with High Glucose

doi: 10.1155/2018/6462793

Figure Lengend Snippet: miR-34a inhibitor significantly suppresses miR-34a expression (by quantitative real-time PCR) stimulated by high glucose (a). And miR-34a inhibitor activates SIRT1/AMPK α and restrains Egr1 expression (by western blotting) in high glucose conditions (b) in RMCs. RMCs were transfected with miR-34a inhibitor (IN) or nonspecific control (N-NC) for 6 hours and further cultured with normal glucose (NG, 5.5 mmol/L glucose) or high glucose (HG, 30 mmol/L glucose) media for 24 hours. ∗ p < 0.05, ∗∗ p < 0.001 versus RMCs cultured with normal glucose media. # p < 0.001 versus RMCs cultured with high glucose media. All results represent means ± SD obtained from three independent experiments in triplicate. And western blotting results were normalized to GAPDH. SIRT1: sirtuin 1; Egr1: early growth response factor 1; p-AMPK α : phosphorylated adenosine monophosphate-activated protein kinase α ; AMPK α : adenosine monophosphate-activated protein kinase α ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Cell Culture

SIRT1 gene silencing suppresses SIRT1 protein (by western blotting) expression in RMCs (a). Transfecting with SIRT1-siRNA obviously decreases phosphorylated AMPK α and increases Egr1 expression (by western blotting) though miR-34a inhibitor in high glucose-cultured RMCs (b). RMCs were transfected with SIRT1-siRNA (S-siRNA), nonspecific control (N-siRNA), or miR-34a inhibitor (IN) for 6 hours and further cultured with normal glucose (NG, 5.5 mmol/L glucose) or high glucose (HG, 30 mmol/L glucose) media for 24 hours. ∗ p < 0.001 versus RMCs cultured with normal glucose media. # p < 0.05, ## p < 0.001 versus RMCs cultured with high glucose media. ¢ p < 0.001 versus RMCs transfected with miR-34a inhibitor. All results represent means ± SD obtained from three independent experiments in triplicate and normalized to GAPDH. SIRT1: sirtuin 1; Egr1: early growth response factor 1; p-AMPK α : phosphorylated adenosine monophosphate-activated protein kinase α ; AMPK α : adenosine monophosphate-activated protein kinase α ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Journal: International Journal of Endocrinology

Article Title: Metformin Regulating miR-34a Pathway to Inhibit Egr1 in Rat Mesangial Cells Cultured with High Glucose

doi: 10.1155/2018/6462793

Figure Lengend Snippet: SIRT1 gene silencing suppresses SIRT1 protein (by western blotting) expression in RMCs (a). Transfecting with SIRT1-siRNA obviously decreases phosphorylated AMPK α and increases Egr1 expression (by western blotting) though miR-34a inhibitor in high glucose-cultured RMCs (b). RMCs were transfected with SIRT1-siRNA (S-siRNA), nonspecific control (N-siRNA), or miR-34a inhibitor (IN) for 6 hours and further cultured with normal glucose (NG, 5.5 mmol/L glucose) or high glucose (HG, 30 mmol/L glucose) media for 24 hours. ∗ p < 0.001 versus RMCs cultured with normal glucose media. # p < 0.05, ## p < 0.001 versus RMCs cultured with high glucose media. ¢ p < 0.001 versus RMCs transfected with miR-34a inhibitor. All results represent means ± SD obtained from three independent experiments in triplicate and normalized to GAPDH. SIRT1: sirtuin 1; Egr1: early growth response factor 1; p-AMPK α : phosphorylated adenosine monophosphate-activated protein kinase α ; AMPK α : adenosine monophosphate-activated protein kinase α ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Techniques: Western Blot, Expressing, Cell Culture, Transfection

Egr1 gene silencing suppresses the Egr1 protein expression (by western blotting) stimulated by high glucose (a). Egr1 gene silencing suppresses the upregulated expression of fibrosis factors (FN and CTGF) (b) (by western blotting) and inflammatory factors (MCP-1 and CXCL5) (c) (by ELISA) stimulated by high glucose in RMCs. RMCs were transfected with nonspecific siRNA (N-siRNA) or three different siRNA targeting Egr1 gene (E1-siRNA, E2-siRNA, and E3-siRNA) for 6 hours and then cultured with high glucose media (30 mmol/L glucose) or normal glucose (NG, 5.5 mmol/L glucose) for 24 hours. ∗ p < 0.05, ∗∗ p < 0.001 versus RMCs cultured with high glucose media. # p < 0.001 versus RMCs cultured with normal glucose media. All results represent means ± SD obtained from three independent experiments in triplicate and normalized to GAPDH. Egr1: early growth response factor 1; FN: fibronectin; CTGF: connective tissue growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MCP-1: monocyte chemoattractant protein 1; CXCL5: chemokine C-X-C motif ligand 5.

Journal: International Journal of Endocrinology

Article Title: Metformin Regulating miR-34a Pathway to Inhibit Egr1 in Rat Mesangial Cells Cultured with High Glucose

doi: 10.1155/2018/6462793

Figure Lengend Snippet: Egr1 gene silencing suppresses the Egr1 protein expression (by western blotting) stimulated by high glucose (a). Egr1 gene silencing suppresses the upregulated expression of fibrosis factors (FN and CTGF) (b) (by western blotting) and inflammatory factors (MCP-1 and CXCL5) (c) (by ELISA) stimulated by high glucose in RMCs. RMCs were transfected with nonspecific siRNA (N-siRNA) or three different siRNA targeting Egr1 gene (E1-siRNA, E2-siRNA, and E3-siRNA) for 6 hours and then cultured with high glucose media (30 mmol/L glucose) or normal glucose (NG, 5.5 mmol/L glucose) for 24 hours. ∗ p < 0.05, ∗∗ p < 0.001 versus RMCs cultured with high glucose media. # p < 0.001 versus RMCs cultured with normal glucose media. All results represent means ± SD obtained from three independent experiments in triplicate and normalized to GAPDH. Egr1: early growth response factor 1; FN: fibronectin; CTGF: connective tissue growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MCP-1: monocyte chemoattractant protein 1; CXCL5: chemokine C-X-C motif ligand 5.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Cell Culture

Metformin reverses the high glucose downregulated SIRT1/AMPK α and upregulated Egr1 protein expression (by western blotting) in RMCs. RMCs were pre-incubated with 50 μ mol/L metformin for 1 hour before stimulating with normal glucose (NG, 5.5 mmol/L glucose) or high glucose (HG, 30 mmol/L glucose) for 24 hours. ∗ p < 0.05, ∗∗ p < 0.001 versus RMCs cultured with normal glucose media. # p < 0.001 versus RMCs cultured with high glucose media. All results represent means ± SD obtained from three independent experiments in triplicate and normalized to GAPDH. Met: metformin; SIRT1: sirtuin 1; Egr1: early growth response factor 1; p-AMPK α : phosphorylated adenosine monophosphate-activated protein kinase α ; AMPK α : adenosine monophosphate-activated protein kinase α ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Journal: International Journal of Endocrinology

Article Title: Metformin Regulating miR-34a Pathway to Inhibit Egr1 in Rat Mesangial Cells Cultured with High Glucose

doi: 10.1155/2018/6462793

Figure Lengend Snippet: Metformin reverses the high glucose downregulated SIRT1/AMPK α and upregulated Egr1 protein expression (by western blotting) in RMCs. RMCs were pre-incubated with 50 μ mol/L metformin for 1 hour before stimulating with normal glucose (NG, 5.5 mmol/L glucose) or high glucose (HG, 30 mmol/L glucose) for 24 hours. ∗ p < 0.05, ∗∗ p < 0.001 versus RMCs cultured with normal glucose media. # p < 0.001 versus RMCs cultured with high glucose media. All results represent means ± SD obtained from three independent experiments in triplicate and normalized to GAPDH. Met: metformin; SIRT1: sirtuin 1; Egr1: early growth response factor 1; p-AMPK α : phosphorylated adenosine monophosphate-activated protein kinase α ; AMPK α : adenosine monophosphate-activated protein kinase α ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Techniques: Expressing, Western Blot, Incubation, Cell Culture

Schematic representation of miR-34a regulating SIRT1/AMPK α signaling pathways and Egr1 involved in the HG-induced fibrosis and inflammation in RMCs. High glucose upregulates miR-34a and Egr1 expression, as well as decreases SIRT1 protein and AMPK α phosphorylation expression. miR-34a suppresses the activation of SIRT1/AMPK α and results in promoting Egr1-mediated inflammation and fibrosis in high glucose-cultured RMCs. AICAR activating AMPK α prevents Egr1 expression in high glucose. Meanwhile, metformin attenuates high glucose-stimulated inflammation and fibrosis in RMCs by regulating miR-34a-mediated SIRT1/AMPK α activity and the downstream Egr1 protein. HG: high glucose; SIRT1: sirtuin 1; AMPK α : adenosine monophosphate-activated protein kinase α ; AICAR: 5-amino-4-imidazolecarboxamide riboside-1-b-D-ribofuranoside; Egr1: early growth response factor 1; FN: fibronectin; CTGF: connective tissue growth factor; MCP-1: monocyte chemoattractant protein 1; CXCL5: chemokine C-X-C motif ligand 5; RMCs: rat mesangial cells.

Journal: International Journal of Endocrinology

Article Title: Metformin Regulating miR-34a Pathway to Inhibit Egr1 in Rat Mesangial Cells Cultured with High Glucose

doi: 10.1155/2018/6462793

Figure Lengend Snippet: Schematic representation of miR-34a regulating SIRT1/AMPK α signaling pathways and Egr1 involved in the HG-induced fibrosis and inflammation in RMCs. High glucose upregulates miR-34a and Egr1 expression, as well as decreases SIRT1 protein and AMPK α phosphorylation expression. miR-34a suppresses the activation of SIRT1/AMPK α and results in promoting Egr1-mediated inflammation and fibrosis in high glucose-cultured RMCs. AICAR activating AMPK α prevents Egr1 expression in high glucose. Meanwhile, metformin attenuates high glucose-stimulated inflammation and fibrosis in RMCs by regulating miR-34a-mediated SIRT1/AMPK α activity and the downstream Egr1 protein. HG: high glucose; SIRT1: sirtuin 1; AMPK α : adenosine monophosphate-activated protein kinase α ; AICAR: 5-amino-4-imidazolecarboxamide riboside-1-b-D-ribofuranoside; Egr1: early growth response factor 1; FN: fibronectin; CTGF: connective tissue growth factor; MCP-1: monocyte chemoattractant protein 1; CXCL5: chemokine C-X-C motif ligand 5; RMCs: rat mesangial cells.

Techniques: Expressing, Activation Assay, Cell Culture, Activity Assay

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