Journal: BMC Developmental Biology

Article Title: Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing

doi: 10.1186/1471-213X-11-28

Figure Lengend Snippet: Luciferase activity in adult Egr-1-luc mice . Transgenic Egr-1-luc mice (4 month old, male or female) were anaesthetized with isofluorane in oxygen and received 6 mg luciferin in 100 μl PBS by i.p. injection. Ten minutes after injection BLI measurement was carried out (1 min signal collection, setting 'high resolution'). A representative animal is shown. The reflected light picture is overlaid by a a liacolor coded BLI image visualized in 'blend mode', which allows allocating the BLI signal to the respective areas shown in the underlying reflected light picture.

Article Snippet: Equal amounts of protein were separated on a 4-20% Tris-glycine gel (Serva) and immunoreactive bands were visualized using Super-Signal-Femto-West (Pierce) with a HRP conjugated rabbit polyclonal antibody against firefly luciferase (1:1000, Santa Cruz Biotechnology), a rabbit monoclonal antibody against Egr-1 (1:500, Cell Signaling) or β-actin (1:2000, Sigma), respectively.

Techniques: Luciferase, Activity Assay, Transgenic Assay, Injection

Journal: BMC Developmental Biology

Article Title: Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing

doi: 10.1186/1471-213X-11-28

Figure Lengend Snippet: Egr-1 promoter driven luciferase activity during postnatal development (day 7 - 21 after birth) . Luciferase activity in Egr-1-luc mice at indicated age was measured by BLI after i.p. injection of luciferin. The luciferase signal was collected for 10 sec from the ventral side of the mice (n = 6). A : A reflected light picture of representative animals at indicated age is overlaid by a color coded BLI image. B and C : Luciferase activity was quantified within regions of interest (ROIs) placed at the paw ( B ) or snout area ( C ) and expressed as photons per second per cm 2 to correct for size differences in ROI size at different ages. A background ROI of similar size was subtracted. Median values of six animals + standard deviation are shown. * p < 0.05, *** p < 0.001; indicated day vs. day 7, Wilcoxon test

Article Snippet: Equal amounts of protein were separated on a 4-20% Tris-glycine gel (Serva) and immunoreactive bands were visualized using Super-Signal-Femto-West (Pierce) with a HRP conjugated rabbit polyclonal antibody against firefly luciferase (1:1000, Santa Cruz Biotechnology), a rabbit monoclonal antibody against Egr-1 (1:500, Cell Signaling) or β-actin (1:2000, Sigma), respectively.

Techniques: Luciferase, Activity Assay, Injection, Standard Deviation

Journal: BMC Developmental Biology

Article Title: Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing

doi: 10.1186/1471-213X-11-28

Figure Lengend Snippet: Immunohistochemical analyses of luciferase and Egr-1 expression during embryonic development . Egr-1-luc ( A , B , C and G ) and wildtype ( D , E and F ) C57BL/6 embryos on day E14 of development were stained for luciferase protein ( A-F ) or Egr-1 protein ( G ). In bone primordia of hindlimbs ( A ), sympathetic paravertebral ganglia ( B ) and masticatory apparatus ( C ) luciferase positive areas (arrows) are stained in lilac in transgenic embryos, in wildtype embryos no luciferase signal was detected ( D-F ). When staining for Egr-1 protein, a similar pattern of protein expression was found - exemplarily shown for the masticatory apparatus ( G ) - as for luciferase ( C ); scale bar: 50 μm

Article Snippet: Equal amounts of protein were separated on a 4-20% Tris-glycine gel (Serva) and immunoreactive bands were visualized using Super-Signal-Femto-West (Pierce) with a HRP conjugated rabbit polyclonal antibody against firefly luciferase (1:1000, Santa Cruz Biotechnology), a rabbit monoclonal antibody against Egr-1 (1:500, Cell Signaling) or β-actin (1:2000, Sigma), respectively.

Techniques: Immunohistochemical staining, Luciferase, Expressing, Staining, Transgenic Assay

Journal: BMC Developmental Biology

Article Title: Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing

doi: 10.1186/1471-213X-11-28

Figure Lengend Snippet: Egr-1 promoter driven luciferase activity after partial liver hepatectomy . A : BLI of sham operated (left) or hepatectomized (right) Egr-1-luc mice at 48 h (top row) and 72 h (bottom row) after surgery. Representative animals from n = 3-6 are shown. Red arrows denote the site of initial surgery, arrowhead points at the edge of a liver lobe showing luciferase activity. B : Quantitative luciferase signals from ROIs placed over the liver area of sham operated or hepatectomized mice 48 h or 72 h after surgery. A representative background ROI of comparable size was subtracted to account for background activity. n = 3-6; mean values of six animals + standard deviation are shown. *p < 0.05 sham vs. hepatectomy (U-test, Mann-Whitney).

Article Snippet: Equal amounts of protein were separated on a 4-20% Tris-glycine gel (Serva) and immunoreactive bands were visualized using Super-Signal-Femto-West (Pierce) with a HRP conjugated rabbit polyclonal antibody against firefly luciferase (1:1000, Santa Cruz Biotechnology), a rabbit monoclonal antibody against Egr-1 (1:500, Cell Signaling) or β-actin (1:2000, Sigma), respectively.

Techniques: Luciferase, Activity Assay, Standard Deviation, MANN-WHITNEY

Journal: BMC Developmental Biology

Article Title: Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing

doi: 10.1186/1471-213X-11-28

Figure Lengend Snippet: Immunohistochemical analyses of Egr-1 driven luciferase expression in regenerating liver . Egr-1-luc mice 4-8 weeks of age were subjected to one third liver hepatectomy as described in "Methods". Forty-eight hours after surgery mice were sacrificed, liver tissue fixed in PFA and stained for luciferase (luc). Tissue next to the site of surgery (rim upper left corner in both images) is shown and cells staining positive for luciferase ( A ) as well as Egr-1 ( B ) appear as clusters with brown-reddish staining (arrows; scale bar: 50 μm)

Article Snippet: Equal amounts of protein were separated on a 4-20% Tris-glycine gel (Serva) and immunoreactive bands were visualized using Super-Signal-Femto-West (Pierce) with a HRP conjugated rabbit polyclonal antibody against firefly luciferase (1:1000, Santa Cruz Biotechnology), a rabbit monoclonal antibody against Egr-1 (1:500, Cell Signaling) or β-actin (1:2000, Sigma), respectively.

Techniques: Immunohistochemical staining, Luciferase, Expressing, Staining

Journal: BMC Developmental Biology

Article Title: Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing

doi: 10.1186/1471-213X-11-28

Figure Lengend Snippet: mRNA and protein levels of Egr-1 and luciferase after partial hepatectomy . Egr-1-luc mice were subjected to one third hepatectomy or sham operation, sacrificed 12 h ( A ) or 48 h ( B ) after surgery and liver tissue subjected to mRNA analyses by qRT-PCR ( A ) or Western blot analyzing protein levels of Egr-1 and luciferase ( B ). A : mRNA levels of Egr-1 and luciferase, respectively (average relative mRNA levels relative to 18S rRNA levels); mRNA levels of luciferase in sham operated animals were below the detection limit. n = 4, mean values + standard deviation are shown; *p < 0.05 sham vs. hepatectomy (U-test, Mann-Whitney). B : Representative Western blots showing the protein levels of Egr-1, luciferase and β-actin for sham operated (left panel) or hepatectomized animals (right panel), respectively; data from two representative animals per treatment are shown. Numbers indicate relative luciferase and Egr-1 expression calculated from optical densities (OD) of luciferase, Egr-1 and ß-actin protein bands. n = 4, mean values + standard deviation are shown; *p < 0.05 sham vs. hepatectomy (U-test, Mann-Whitney).

Article Snippet: Equal amounts of protein were separated on a 4-20% Tris-glycine gel (Serva) and immunoreactive bands were visualized using Super-Signal-Femto-West (Pierce) with a HRP conjugated rabbit polyclonal antibody against firefly luciferase (1:1000, Santa Cruz Biotechnology), a rabbit monoclonal antibody against Egr-1 (1:500, Cell Signaling) or β-actin (1:2000, Sigma), respectively.

Techniques: Luciferase, Quantitative RT-PCR, Western Blot, Standard Deviation, MANN-WHITNEY, Expressing

Journal: BMC Developmental Biology

Article Title: Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing

doi: 10.1186/1471-213X-11-28

Figure Lengend Snippet: In vivo bioluminescence imaging of the ear wound . In Egr-1-luc mice between the ages of 4-8 weeks an ear wound was inflicted in one ear. Twenty-four hours after infliction animals were subjected to BLI. Luciferase signal was collected for 2 min from the wounded or the control ear, respectively. A : Color coded BLI image overlaid onto a reflected light image from the control ear (left) or wounded ear (right) immediately (top row) or 24 h (bottom row) after wound infliction. B : Quantitative luciferase signals from ROIs placed over the wound site or a similar sized ROI at the control ear. n = 3, mean values + standard deviation are shown; *p < 0.05 control vs. wound (U-test, Mann-Whitney).

Article Snippet: Equal amounts of protein were separated on a 4-20% Tris-glycine gel (Serva) and immunoreactive bands were visualized using Super-Signal-Femto-West (Pierce) with a HRP conjugated rabbit polyclonal antibody against firefly luciferase (1:1000, Santa Cruz Biotechnology), a rabbit monoclonal antibody against Egr-1 (1:500, Cell Signaling) or β-actin (1:2000, Sigma), respectively.

Techniques: In Vivo, Imaging, Luciferase, Standard Deviation, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

doi: 10.1371/journal.pone.0115170

Figure Lengend Snippet: (A) HMEC-1s and HUVECs were incubated with SW480-derived EVs (1 µg/mL) or untreated control. mRNA was isolated from untreated control cells or cells treated with EVs for 0, 0.5, 1, 2, and 4 h and analyzed using real time RT-PCR (n = 3). Values represent Egr-1 mRNA/GAPDH mRNA normalized to untreated control cells. (B) HMEC-1s were treated with EVs (1 µg/mL) derived from HCT116 colorectal carcinoma, A549 lung adenocarcinoma, HT1080 fibrosarcoma, PC3 prostate carcinoma, SH-SY5Y neuroblastoma, and BEAS-2B normal bronchial epithelial cells for 0.5 h (n = 3). (C, D) In HMEC-1s, nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 0.5, 1, 2, and 4 h was analyzed under confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel C. Scale bars represent 30 µm. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over that of total cells (D). Data are represented as mean ± SD. * P <0.05; ** P <0.01; ** P <0.001; n.s., not significant.

Article Snippet: Cells were treated with SW480-derived EVs (1 µg/mL, 0.5 mL), fixed with 4% paraformaldehyde, and incubated with rabbit anti-Egr-1 antibody (Cell Signaling Technology, Hitchin, United Kingdom) followed by AlexaFluor488 goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Incubation, Derivative Assay, Isolation, Quantitative RT-PCR, Translocation Assay, Confocal Microscopy, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

doi: 10.1371/journal.pone.0115170

Figure Lengend Snippet: (A) HMEC-1s were transfected with 50 nM of scrambled siRNA or Egr-1 siRNA-1, Egr-1 siRNA-2, or Egr-1 siRNA-3. mRNAs were isolated from the cells after 48 h and the level of Egr-1 mRNA was analyzed using real time RT-PCR (n = 3). (B) Nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 1 h was analyzed in scrambled siRNA (50 nM) or Egr-1 siRNA-1 (50 nM) transfected HMEC-1s (n = 3). (C, D) Confluent HMEC-1s transfected with scrambled siRNA (50 nM) or Egr-1 siRNA-1 (50 nM) were scratched and treated with SW480-derived EVs (1 µg/mL); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). The number of migrated cells in the denuded zone of each group is shown in panel D. Scale bars represent 100 µm. Data are represented as mean ± SD. * P <0.05; ** P <0.01; *** P <0.001; n.s., not significant.

Article Snippet: Cells were treated with SW480-derived EVs (1 µg/mL, 0.5 mL), fixed with 4% paraformaldehyde, and incubated with rabbit anti-Egr-1 antibody (Cell Signaling Technology, Hitchin, United Kingdom) followed by AlexaFluor488 goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Transfection, Isolation, Quantitative RT-PCR, Translocation Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

doi: 10.1371/journal.pone.0115170

Figure Lengend Snippet: (A, B) HMEC-1s were pretreated with signaling inhibitors for 1 h and then stimulated for 1 h with SW480-derived EVs (1 µg/mL). Nuclear translocation of Egr-1 protein was analyzed using confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel A. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over total cells (B). (C, D) Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL) in the presence or absence of signaling inhibitors; then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). Representative photographs of confocal microscopic imaging are shown in panel C and the number of migrated cells in the denuded zone of each group is shown in panel D. ERK1/2 inhibitor, PD98059 (20 µM); p38 MAPK inhibitor, SB203580 (10 µM); JNK inhibitor, SP600125 (20 µM); Akt inhibitor, BML-257 (20 µM). (E, F) C57BL/6 mice were subcutaneously injected with Matrigel containing SW480-derived EVs (20 µg) with PD98059 (20 µM) or SP600125 (20 µM). After 7 days, whole-mount staining of Matrigel with anti-CD31 antibody was conducted (n = 5). Representative confocal Z-stack photographs of whole mounts stained for CD31 (green) are shown in panel E. Fluorescence intensities of CD31 in the Z-stack plane of the Matrigel were measured as described in the (F). Scale bars in panels A, C, and E represent 30, 100, and 100 µm, respectively. Data are represented as mean ± SD. *** P <0.001.

Article Snippet: Cells were treated with SW480-derived EVs (1 µg/mL, 0.5 mL), fixed with 4% paraformaldehyde, and incubated with rabbit anti-Egr-1 antibody (Cell Signaling Technology, Hitchin, United Kingdom) followed by AlexaFluor488 goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Derivative Assay, Translocation Assay, Confocal Microscopy, Staining, Fluorescence, Imaging, Injection

Journal: PLoS ONE

Article Title: Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

doi: 10.1371/journal.pone.0115170

Figure Lengend Snippet: (A) HMEC-1s were treated with DiI-labeled SW480-derived EVs (1 µg/mL) for 1 h in the presence or absence of MβCD (10 mM) (n = 3). SW480-derived EVs and nuclei were stained with DiI (red) and Hoechst (blue) respectively. Representative photographs are shown in panel A. Scale bars represent 10 µm. (B) Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL) in the presence or absence of MβCD (10 mM); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). (C, D) HMEC-1s were pretreated with MβCD (10 mM) for 1 h and then stimulated with SW480-derived EVs (1 µg/mL) for 1 h. Scale bars represent 30 µm. Nuclear translocation of Egr-1 protein was analyzed using confocal microscopy and the percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus co-localized signals over that of total cells (n = 3). Data are represented as mean ± SD. *** P <0.001.

Article Snippet: Cells were treated with SW480-derived EVs (1 µg/mL, 0.5 mL), fixed with 4% paraformaldehyde, and incubated with rabbit anti-Egr-1 antibody (Cell Signaling Technology, Hitchin, United Kingdom) followed by AlexaFluor488 goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Labeling, Derivative Assay, Staining, Translocation Assay, Confocal Microscopy

Journal: PLoS ONE

Article Title: IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM

doi: 10.1371/journal.pone.0039811

Figure Lengend Snippet: Growth-quiescent SMCs were treated with MMP inhibitor GM6001+ (25 µM) and TAPI-1 (10 µM) for 30 min before stimulation with IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) Egr-1 mRNA levels in GM6001+ or GM6001- treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs treated with GM6001+ or GM6001-. ( C ) Egr-1 mRNA levels in TAPI-1 treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( D ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with TAPI-1. DMSO was used as a carrier. ( E ) Western blotting for ADAM17, ADAM10, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ADAM17 siRNA (0.1 µM) and treated with IL-1beta for 30 min.

Article Snippet: Rabbit monoclonal antibodies Egr-1 were obtained from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Transfection

Journal: PLoS ONE

Article Title: IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM

doi: 10.1371/journal.pone.0039811

Figure Lengend Snippet: Growth-quiescent SMCs were incubated with EGFR inhibitors AG1478 (5 µM) and PD153035 (5 µM) for 30 min, and exposed to IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) Egr-1 mRNA levels in AG1478- and PD153035-treated SMCs incubated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with AG1478 or PD153035. ( C ) Western blotting for EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with EGFR siRNA (0.1 µM) and treated with IL-1beta for 30 min. ( D ) Western blotting for ErbB4, EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ErbB4 siRNA (0.1 µM) and treated with IL-1beta for 30 min. ( E ) Egr-1 mRNA levels in AG825 (10 µM for 30 min)-treated SMCs incubated with IL-1beta for 30 min by qPCR. Data were normalized to beta-actin. ( F ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs pretreated with AG825 (10 µM for 30 min) then stimulated with IL-1beta for another 30 min.

Article Snippet: Rabbit monoclonal antibodies Egr-1 were obtained from Cell Signaling (Danvers, MA, USA).

Techniques: Incubation, Western Blot, Transfection

Journal: PLoS ONE

Article Title: IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM

doi: 10.1371/journal.pone.0039811

Figure Lengend Snippet: ( A ) Growth-quiescent SMCs were stimulated with IL-1beta (10 ng/ml) for the indicated times. Total protein extracts were resolved by SDS-PAGE and subjected to Western blot analysis using antibodies for Egr-1, EGFR phospho-Tyr 845 , total EGFR and beta-actin. Alternatively, Western blotting for EGFR phospho-Tyr 845 or EGFR of total extracts of SMCs treated with ( B ) AG1478 (5 µM), ( C ) GM6001+ or GM6001- (25 µM) ( D ) TAPI-1 (10 µM), PD153035 (5 µM) for 30 min followed by IL-1beta stimulation for 5 min.

Article Snippet: Rabbit monoclonal antibodies Egr-1 were obtained from Cell Signaling (Danvers, MA, USA).

Techniques: SDS Page, Western Blot

Journal: PLoS ONE

Article Title: IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM

doi: 10.1371/journal.pone.0039811

Figure Lengend Snippet: ( A ) Western blotting for Egr-1, ADAM17, EGFR, IL-1RI or beta-actin using total extracts of growth-quiescent wild-type or ADAM17-deficient mEFs treated with IL-1beta for 30 or 60 min. ( B ) Interaction of 125 I-IL-1beta with ADAM17WT and ADAM17-deficient cells. Growth-quiescent ADAM17WT and ADAM17−/− mEFs (1.8×10 4 cells/well) were incubated with increasing amounts of 125 I-IL-1beta in 1% BSA/PBS for 1 h at 4°C. The cells were washed and lysed with 1M NaOH prior to assessment of counts in an automated gamma-counter.

Article Snippet: Rabbit monoclonal antibodies Egr-1 were obtained from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Incubation

Journal: Theranostics

Article Title: Loss of EGR-1 uncouples compensatory responses of pancreatic β cells

doi: 10.7150/thno.40664

Figure Lengend Snippet: EGR-1 deficiency exhibits reduced proliferation and neogenesis in the islet upon HF feeding. A : Immunofluorescence staining for Ki67 ( green ) and B : quantification of Ki67 + cells in the pancreas of 5-month-old male WT ( n =4) and Egr1 -/- ( n =4) mice fed a HF diet for 3 months. Numbers inside bars are accumulated percentages of different categories of Ki67 + cell number per islet. C : Quantification of Ki67-positive insulin-positive cells. Scale bar: 50 μm. D : Immunofluorescence staining for cleaved caspase-3 ( green ) in the pancreatic islets. Images are co-stained for insulin ( red ) and nuclei ( blue ). Scale bar: 200 μm. E : Quantification of caspases-3-positive insulin-positive cells. F : mRNA levels of proliferation markers in the isolated islets of HF-fed Egr1 -/- ( n =10) mice relative to WT ( n =6) mice. G : Immunoblot analysis on proliferation-related markers and signaling molecules. The relative intensities of the bands by densitometric quantification to WT are indicated. * P <0.05 and *** P <0.001.

Article Snippet: EGR-1 antibody (#4154S; Cell signaling) was used to immunoprecipitate the EGR-1 protein and DNA complexes.

Techniques: Immunofluorescence, Staining, Isolation, Western Blot

Journal: Theranostics

Article Title: Loss of EGR-1 uncouples compensatory responses of pancreatic β cells

doi: 10.7150/thno.40664

Figure Lengend Snippet: Decreased transcriptional network in the islets of HF-fed Egr1 -/- mice. A : Bioinformatics analysis in identification of transcriptional factors and endocrine molecules that contain potential EGR-1 binding site. Sequences for proteins involved in the development program of islet lineages were analyzed for potential EGR-1 binding site using the PROMO transcription factor binding site database. B : Expression of genes for pancreatic development, endocrine lineage specification and β-cell identity in the isolated islets of HF-fed Egr1 -/- ( n =10) relative to WT ( n =6) mice. * P <0.05 and ** P <0.01 compared to WT mice. Pathway analysis of the response of HF feeding on the islets of ( C ) WT and ( D ) Egr1 -/- mice using Ingenuity Pathways Analysis (IPA) software. Pathway analysis of the effect of EGR-1 deficiency on the transcription network in ( E ) RC and ( F ) HF diet feeding. Color shading corresponds to the type of dysregulation: red for up-regulated and green for down-regulated genes according to the results of quantitative PCR. The shape of the node indicates the major function of the protein: transcription regulators (dumbbell); kinases (three arrows); and others (circle). G : Expression of genes in EGR-1 knockdowned ( n =5) relative to control ( n =5) MIN6 cells. H : ChIP assay in EGR-1 overexpressed and control (pCMV) MIN6 cells. Sequences containing the potential EGR-1 binding sites in Pdx1 and Arx were amplified by real-time PCR. n =3 in each group. * P <0.05, ** P <0.01, and *** P <0.001.

Article Snippet: EGR-1 antibody (#4154S; Cell signaling) was used to immunoprecipitate the EGR-1 protein and DNA complexes.

Techniques: Binding Assay, Expressing, Isolation, Software, Real-time Polymerase Chain Reaction, Amplification

Journal: Theranostics

Article Title: Loss of EGR-1 uncouples compensatory responses of pancreatic β cells

doi: 10.7150/thno.40664

Figure Lengend Snippet: The correlation between EGR-1 expression and compensatory genes in human pancreatic tissues. Scatter plot illustrating the Spearman's correlation of normalized reads per patient between EGR1 and PDX1 , as well as compensatory genes, such as CCND1 (cyclin D1), INS (insulin), and GCK (glucokinase), in ( A ) non-diabetic (upper panels) and diabetic (lower panels) subjects; and ( B ) normal weight (BMI < 23, upper panels) and overweight/obese (BMI ≥ 23, lower panels) subjects. Spearman's rank correlation coefficients r and P value are provided in each plot. * P <0.05, ** P <0.01, and *** P <0.001.

Article Snippet: EGR-1 antibody (#4154S; Cell signaling) was used to immunoprecipitate the EGR-1 protein and DNA complexes.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Xanthine Oxidase-Derived ROS Upregulate Egr-1 via ERK1/2 in PA Smooth Muscle Cells; Model to Test Impact of Extracellular ROS in Chronic Hypoxia

doi: 10.1371/journal.pone.0027531

Figure Lengend Snippet: Egr-1 mRNA expression was measured by qPCR in the pulmonary artery of 2 week old chronically hypoxic calves compared to age-matched normoxic control calves . Data are expressed as copies of Egr-1 per copy HPRT, mean ± SEM. *p<0.05 vs. Norm; n = 4–5.

Article Snippet: Blots from human PASMC total cell lysates were also probed with polyclonal rabbit anti-Egr-1 (Cell Signaling, 1∶500), cyclin D1 (Cell Signaling , 1∶1,000) and ß-actin as a loading control.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Xanthine Oxidase-Derived ROS Upregulate Egr-1 via ERK1/2 in PA Smooth Muscle Cells; Model to Test Impact of Extracellular ROS in Chronic Hypoxia

doi: 10.1371/journal.pone.0027531

Figure Lengend Snippet: ( A ) Egr-1 mRNA expression was measured by qPCR in PASMC exposed to xanthine oxidase (XO- 8 mU/mL) and hypoxanthine (0.5 mM) at 37°C for 1 hour (XO). Data are expressed as fold change in Egr-1/HPRT relative to vehicle treated cells and presented as mean ± SEM. *p<0.05 vs. vehicle treated cells. B. Extracellular O 2 .− was measured by the SOD-inhibitable reduction of XTT in XO+hypoxanthine-treated (XO), and vehicle-treated smooth muscle cells with and without SOD (500 U/mL). 20,000 cells were treated in a final volume of 150 µL and exposed to XTT for 1 hour at 37°C and pH 7.4. An extinction coefficient of 21600 M −1 cm −1 (XTT) was used to calculate O 2 .− flux and data expressed in M•s −1 . *p<0.05 vs. vehicle-treated cells, n = 3. C. H 2 O 2 was measured with a standard fluorometric assay utilizing in which H 2 O 2 reacts with HRP to forms compound I, which subsequently oxidizes p HPA to its dimer form which can be detected on a fluorometer. Experiments were performed with 30,000 cells per well and a final volume of 150 µL for 2 hours at 37°C and pH 7.4 and H 2 O 2 flux expressed in M•s −1 . *p<0.05 vs. vehicle-treated cells, n = 3. Data expressed as mean ± SEM.

Article Snippet: Blots from human PASMC total cell lysates were also probed with polyclonal rabbit anti-Egr-1 (Cell Signaling, 1∶500), cyclin D1 (Cell Signaling , 1∶1,000) and ß-actin as a loading control.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Xanthine Oxidase-Derived ROS Upregulate Egr-1 via ERK1/2 in PA Smooth Muscle Cells; Model to Test Impact of Extracellular ROS in Chronic Hypoxia

doi: 10.1371/journal.pone.0027531

Figure Lengend Snippet: A. Egr-1 mRNA expression in calf PASMC exposed in sealed humidified gas chambers with 21% or 1% O 2 plus 5% CO 2 and balanced N 2 at 37°C for 4 and 24 hours and shown compared to XO/HX treatment for 1 hour (XO). B. Extracellular O 2 .− production in PASMC exposed to XO/HX (XO) or hypoxia as described above. 20,000 cells treated in a final volume of 150 µL were exposed to XO/HX (XO) for 1 or 4 hours or hypoxia for 4 hours at 37°C and pH 7.4 in the presence of XTT. An extinction coefficient of 21600 M −1 cm −1 was used to calculate O 2 .− flux (M-s −1 ). C. Egr-1 mRNA expression in calf PASMC exposed to antimycin A to increase endogenous ROS generated in the mitochondria. 20,000 cells were exposed to 0.25 µM or 250 µM antimycin A for 2 hours at 37°C. All data expressed as mean ± SEM. *p<0.05 vs. vehicle-treated cells, n = 3.

Article Snippet: Blots from human PASMC total cell lysates were also probed with polyclonal rabbit anti-Egr-1 (Cell Signaling, 1∶500), cyclin D1 (Cell Signaling , 1∶1,000) and ß-actin as a loading control.

Techniques: Expressing, Generated

Journal: PLoS ONE

Article Title: Xanthine Oxidase-Derived ROS Upregulate Egr-1 via ERK1/2 in PA Smooth Muscle Cells; Model to Test Impact of Extracellular ROS in Chronic Hypoxia

doi: 10.1371/journal.pone.0027531

Figure Lengend Snippet: Calf PASMC were treated with XO (8 mU/mL) and hypoxanthine (0.5 mM) for 1 hour (XO) and protein was isolated for Western blot analysis. A. Blots were probed with antibodies against phosphorylated ERK1/2 (pERK) and total ERK1/2 (tERK) Each panel shows representative lanes from a single immunoblot as well as the densitometry data representing the ratio of the phosphorylated to total ERK1/2. B. Calf PASMC were pretreated with the MAPK/ERK1/2 inhibitor PD98059 (10 µM) for 30 min followed by either XO/HX (XO) or vehicle control. mRNA was isolated and analyzed by qPCR for Egr-1 expression relative to HPRT. C. Calf PASMC were pretreated with CuZn SOD (500 U/mL) and catalase (600 U/mL) for 30 minutes prior to XO (8 mU/mL) and hypoxanthine (0.5 mM). Total protein was subjected to Western blot analysis for phosphorylated ERK1/2 (pERK) and total ERK1/2 (tERK). The band intensities were quantified by densitometry and normalized to that of vehicle. D. PASMC were pretreated with CuZn SOD (500 U/mL) and catalase (600 U/mL) for 30 minutes prior to XO (8 mU/mL) and hypoxanthine (0.5 mM). mRNA was isolated and analyzed by qPCR for Egr-1 expression relative to HPRT. All data expressed as mean ± SEM. *p<0.05 vs. vehicle treated cells; # p<0.05 vs. 1 hour XO-treated cells; n = 3.

Article Snippet: Blots from human PASMC total cell lysates were also probed with polyclonal rabbit anti-Egr-1 (Cell Signaling, 1∶500), cyclin D1 (Cell Signaling , 1∶1,000) and ß-actin as a loading control.

Techniques: Isolation, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Xanthine Oxidase-Derived ROS Upregulate Egr-1 via ERK1/2 in PA Smooth Muscle Cells; Model to Test Impact of Extracellular ROS in Chronic Hypoxia

doi: 10.1371/journal.pone.0027531

Figure Lengend Snippet: A. PASMC isolated from three chronically hypoxic calves and three normoxic age-matched control calves were treated simultaneously with xanthine oxidase (8 mU/mL) and hypoxanthine (0.5 mM) at 37°C for 1 hour (XO). Egr-1 mRNA expression was measured by qPCR and expressed as fold change in Egr-1/HPRT relative to the vehicle-treated PASMC isolated from normoxic calves. Data are presented as mean ± SEM. *p<0.05 vs. vehicle treated cells. B. For protein analysis, PASMC (1×10 6 cells per plate) isolated from a chronically hypoxic calf and a normoxic age-matched control calf were treated simultaneously with xanthine oxidase (8 mU/mL) and hypoxanthine (0.5 mM) at 37°C for 1 hour (XO) with and without a 30 minute pretreatment with either Cu,Zn SOD (500 U/mL) and catalase (600 U/mL) or with the MAPK/ERK1/2 inhibitor PD98059 (10 µM). Nuclear protein was isolated using Thermo-Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagents, and 25 µg was loaded on a gel for Western blot analysis with the Egr-1 antibody (1∶1,000). Ponceau S staining was performed on the gel to confirm protein equal loading. (see ).

Article Snippet: Blots from human PASMC total cell lysates were also probed with polyclonal rabbit anti-Egr-1 (Cell Signaling, 1∶500), cyclin D1 (Cell Signaling , 1∶1,000) and ß-actin as a loading control.

Techniques: Isolation, Expressing, Western Blot, Staining

Journal: PLoS ONE

Article Title: Xanthine Oxidase-Derived ROS Upregulate Egr-1 via ERK1/2 in PA Smooth Muscle Cells; Model to Test Impact of Extracellular ROS in Chronic Hypoxia

doi: 10.1371/journal.pone.0027531

Figure Lengend Snippet: Human PASMC were transfected with human EC-SOD siRNA (siEC-SOD) or the negative control siRNA vector (siNEG) and experiments were performed 48 hours later. A. EC-SOD mRNA expression expressed relative to GAPDH to test the extent of knock-down of EC-SOD with siRNA. B. Fold change in Egr-1 mRNA expression expressed relative to GAPDH in human PASMC treated with siEC-SOD compared to cells treated with siNEG. Data are presented as mean ± SEM. *p<0.05 vs. siNEG. C. Western blot analysis in total cell lysates from human PASMC treated with siEC-SOD or siNEG. The blots were probed sequentially with the human EC-SOD antibody (1∶1,000), Egr-1 antibody (1∶500) cyclin D1 and ß-actin (1∶1,000) to confirm equal protein loading. Blots were also probed with the phosphorylated ERK1/2 and total ERK1/2 antibodies (1∶1,000). Densitometry shows fold change in EC-SOD, Egr-1 or cyclin D1 relative to ß-actin and fold change in phosphorylated relative to total ERK1/2 (n = 2). D. Cell proliferation measured by MTS assay. Experiments were done in triplicate. *p<0.05 vs. siNEG. Solid line (▪): siNEG; dashed line (▴): siEC-SOD *p<0.05 vs. siNEG E . Apoptosis was assayed using the Guava Nexin kit (Guava Technologies) according to the manufacturer's protocol. Fluorescence emission for Annexin V staining and 7-AAD was measured by flow cytometry. Experiments were done in triplicate. *p<0.05 vs. siNEG.

Article Snippet: Blots from human PASMC total cell lysates were also probed with polyclonal rabbit anti-Egr-1 (Cell Signaling, 1∶500), cyclin D1 (Cell Signaling , 1∶1,000) and ß-actin as a loading control.

Techniques: Transfection, Negative Control, Plasmid Preparation, Expressing, Western Blot, MTS Assay, Fluorescence, Staining, Flow Cytometry

Journal: Experimental Hematology & Oncology

Article Title: Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma

doi: 10.1186/2162-3619-2-4

Figure Lengend Snippet: Kinetics of BCR-induced expressions of EGR-1 and c-MYC in MCL cell lines and primary MCL cells. ( A ) Kinetics of BCR-induced mRNA expression of EGR-1 and c-MYC in Granta-519 and HBL-2 cell lines. 3×10 6 cells/ml were stimulated with 10 μg/ml of immobilized anti-IgM antibody for 15 min, 30 min, 1 h, 3 h and 6 h. EGR-1 and c-MYC expressions were analyzed by qRT-PCR. ( B ) Induction of EGR-1 protein upon BCR engagement was confirmed by western-blot. ( C ) The same experiments were performed on primary patients’ cells. EGR-1 and c-MYC expressions were analyzed by qRT-PCR (left panel) and western-blot (right panels). ( D ) EGR-1 and c-MYC mRNA expressions upon anti-IgM stimulation (1 h) were analyzed by qRT-PCR from 7 patients’ samples (UPN2, 3, 4, 5, 6, 7 and 8). Fold increase of mRNA level were calculated compared with unstimulated cells in all experiments. All measurements were done in duplicate and the mean is provided.

Article Snippet: Antibodies to EGR-1 (mAb 44D5), c-MYC (mAb 9B11), phospho-Src family (mAb 100 F9) also reactive with phospho-Tyr397 LYN (catalytic site) and phospho-JNK (Thr183/Tyr185) were from Cell Signaling (Beverley, MA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Experimental Hematology & Oncology

Article Title: Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma

doi: 10.1186/2162-3619-2-4

Figure Lengend Snippet: Targeting JNK and EGR-1 induces MCL apoptosis and decreases BCR-induced cell survival. ( A ) HBL-2 and Granta-519 cells were treated with SP600125 for 48 h and apoptosis was measured by flow cytometry. Percentage of apoptotic cells corresponded to% of annexin V-positive, including PI-negative and PI-positive cells. Mean ± SD of 2 independent experiments is represented. ( B ) Patients’ cells (UPN1, 2, 8 and 11) were stimulated with immobilized anti-IgM for 24 h with or without SP600125 (10 μM) and the percentage of apoptotic cells was determined by flow cytometry after gating on CD19+ cells ( left panel ). All measurements were done in duplicate and the mean is provided. Results are also shown as median ± quartile ( box ) ± SE ( bars ) (n=4) ( right panel ). Differences between groups were determined using the paired Student t test. ( C ) Jeko-1 cells were transfected either with control siRNA (Ctrl1) or EGR-1 siRNA and EGR1 protein level was determined by western-blot after 72 h of culture ( left panel ). Primary MCL cells (UPN9) were transfected either with controls siRNA (Ctrl1, Ctrl2) or EGR-1 siRNA and subsequently stimulated with anti-IgM for 24 h or left unstimulated ( right panel ). Percentage of apoptotic cells was normalized to unstimulated cells and calculated as follows: [(% apoptosis BCR stimulated cells -% apoptosis BCR-unstimulated cells) / (% apoptosis BCR-unstimulated cells)] x100. All measurements were done in duplicate and the mean value is provided.

Article Snippet: Antibodies to EGR-1 (mAb 44D5), c-MYC (mAb 9B11), phospho-Src family (mAb 100 F9) also reactive with phospho-Tyr397 LYN (catalytic site) and phospho-JNK (Thr183/Tyr185) were from Cell Signaling (Beverley, MA).

Techniques: Flow Cytometry, Transfection, Western Blot

Journal: Experimental Hematology & Oncology

Article Title: Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma

doi: 10.1186/2162-3619-2-4

Figure Lengend Snippet: PP2 and dasatinib inhibit BCR-induced LYN and JNK activation and EGR-1 upregulation. ( A ) Patients’ cells (UPN9) were pretreated with dasatinib (100nM) or SP600125 (10 μM) for 1 h and stimulated for 5 min or 15 min with soluble anti-IgM (10 μg/ml). Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. ( B ) The same experiment was done with PP2 (10 μM) on UPN 9 and UPN 13 under the same conditions of BCR stimulation for 10 min. Lines 1 and 2 have to be compared to evidence the effect of PP2 on the constitutive level of phosphorylation for Lyn. Similarly lines 3 and 4 reflect this effect upon BCR stimulation. ( C ) BCR-induced phospho-JNK (p54 and p46) was analyzed under treatment with dasatinib (100nM) or SP600125 (10 μM) used herein as a positive control of phospho-JNK inhibition. ( D ) Impact of dasatinib on BCR-induced EGR-1 expression. MCL cells were pretreated with various concentrations of dasatinib as indicated and stimulated with immobilized anti-IgM. EGR-1 mRNA and protein were analyzed by qRT-PCR at 1 h of stimulation ( left panel ) and western-blot at 3 h of stimulation ( right panel ). Relative mRNA expression was calculated compared with unstimulated cells.

Article Snippet: Antibodies to EGR-1 (mAb 44D5), c-MYC (mAb 9B11), phospho-Src family (mAb 100 F9) also reactive with phospho-Tyr397 LYN (catalytic site) and phospho-JNK (Thr183/Tyr185) were from Cell Signaling (Beverley, MA).

Techniques: Activation Assay, Positive Control, Inhibition, Expressing, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Hippocampal cAMP-dependent synaptic potentiation, ERK-dependent immediate-early gene activation, and context-dependent fear conditioning in mice are linked through dependence on the guanine nucleotide exchange factor RapGEF2

doi: 10.1101/2022.04.30.490162

Figure Lengend Snippet: (A) Representative images of cFos immunostaining in hippocampal CA1, DG and CA3 and basolateral amygdala of flox and cKO mice that were sacrificed 1hr after fear conditioning (FC) or stayed in the home cage (NFC). Scale bar: 200 μm (B) Quantification of cFos IR in hippocampal subregions and basolateral amygdala of flox and cKO mice indicated that fear-conditioning induced cFos increase in all these regions. No significant difference was observed between flox and cKO mice. N=3~6 for each group. Two-way ANOVA following by post hoc Bonferroni t-test, *p<0.05, **p<0.001. (C) Representative images of Egr-1 immunostaining in hippocampal CA1, DG and CA3 and basolateral amygdala of flox and cKO mice after fear conditioning. Scale bar: 200 μm (D) Quantification of Egr-1 IR indicated that fear-conditioning induced Egr-1 increase in CA1 and DG is RapGEF2-dependent. cKO mice with RapGEF2 ablation in CA1 and DG showed attenuated Egr-1 increase in CA1 and DG 1hr after fear conditioning, compared to flox mice. N=3~6 for each group. Two-way ANOVA following by post hoc Bonferroni t-test, *p<0.05, **p<0.001. (E) RNAscope with egr-1 (in red) and c-fos (in green) probes indicated that upregulation of c-fos mRNA in hippocampal CA1 and DG following fear conditioning occurred exclusively in the neurons with upregulation of egr-1 mRNA. In CA1, 67.77% ± 10.92% of Egr-1 positive neurons are c-fos positive. In DG, 87.89% ± 7.67% of egr-1 positive neurons are cfos positive. Scale bar: 100 μm (left panels), 20 μm (right panels).

Article Snippet: The primary antibodies used were rabbit anti-pERK (1:1500, Cell Signaling Technology, Danvers, MA), anti-c-Fos (1:5000, EnCor Biotechnology Inc., Gainesville, FL), anti-Egr-1 (15F7) (1:1000, Cell Signaling Technology, Danvers, MA) and rabbit anti-Rapgef2 (NNLE-2, custom-made by Anaspec, see ref. ( )).

Techniques: Immunostaining