Journal: bioRxiv

Article Title: Inverse Agonist Activity of Angiotensin II Receptor Blocker Is Crucial for Prevention of Aortic Aneurysm Formation in Marfan Syndrome

doi: 10.1101/2022.12.21.521508

Figure Lengend Snippet: Immunoblot analysis of Egr-1 protein in ascending aorta of 16-week-old vehicle-treated Fbn1 +/+ mice and Fbn1 C1041G/+ mice treated with vehicle, candesartan cilexetil (Can) (1 mg/kg/day), candesartan-7H (Can-7H) (1 mg/kg/day), or Can-7H (20 mg/kg/day). The intensity of each band was quantified by densitometric analysis and corrected for the amount of Gapdh protein as an internal control (n = 5–9) (mean ± SEM). * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The membranes were blocked with 1% bovine serum albumin (Sigma-Aldrich) or 5% skim milk powder (FUJIFILM Wako Pure Chemical Corporation) in TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20), and incubated with the following primary antibodies: rabbit monoclonal anti-phosphorylated Smad2 (Ser465/467) antibody (Cell Signaling Technology, #3108), rabbit monoclonal anti-Smad2 antibody (Cell Signaling Technology, #5339), rabbit polyclonal antiphosphorylated ERK1/2 (Thr202/Tyr204) antibody (Cell Signaling Technology, #9101), rabbit polyclonal anti-ERK1/2 antibody (Cell Signaling Technology, #9102), rabbit monoclonal anti-EGR1 antibody (Cell Signaling Technology, #4154), and rabbit monoclonal anti-GAPDH antibody (Cell Signaling Technology, #2118).

Techniques: Western Blot

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: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

doi: 10.1371/journal.pone.0042717

Figure Lengend Snippet: HGF-induced activation of Egr1 mRNA and protein level ( 2A, 2C ) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting ( 2B, 2D ). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity ( 2E, 2F ). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Construct

Journal: PLoS ONE

Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

doi: 10.1371/journal.pone.0042717

Figure Lengend Snippet: HGF-induced activation of Egr1 and the effect of c-Met inhibition on HGF-induced Egr1 expression in SK-HEP-1 cells were examined by immunoblotting and luciferase reporter assays ( 5A, 5B ). Transcriptional activity of Egr1 was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with a pGL2-Luc-B-Egr1 plasmid construct and pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity. Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Inhibition, Expressing, Western Blot, Luciferase, Activity Assay, Reporter Assay, Transfection, Plasmid Preparation, Construct

Journal: PLoS ONE

Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

doi: 10.1371/journal.pone.0042717

Figure Lengend Snippet: To achieve an inducible knockdown of Egr1, we firstly transfected SK-HEP-1 cells with pCDNA6/TR which express tet repressor. Stable cells were selected using Blasticidin before co-transfecting SK-HEP-1 T-REx cells with tetracycline–regulated pSUPER.retro.neo+GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were selected using G418. After selection, clones were analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold down-regulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones ( 6A ) but not in mock transfected clones ( 6B ). The graph compares the signal intensities obtained from Egr1 bands.

Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Construct, Sequencing, Selection, Clone Assay, Western Blot

Journal: PLoS ONE

Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

doi: 10.1371/journal.pone.0042717

Figure Lengend Snippet: SK-HEP-1 TREx cells were obtained by stable transfection of pCDNA-6/TR. Then SK-HEP-1 TREx cells were transfected with recombinant pSUPER-retro.neo+GFP vector expressing Egr-1 targeted shRNA or empty pSUPER-retro.neo+GFP vector using Fugene as described in . Knockdown of Egr1 in Egr1-shRNA-expressing SK-HEP-1 stable cell lines were induced by tetracycline ( 7A ). Equal amount of tetracycline induced and un-induced Egr1-shRNA-expressing SK-HEP-1 cells were used for migration and invasion assays in the presence and absence of HGF ( 7B, 7C ). Results are expressed as fold differences of matrigel invaded cells and are representative of two or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. Zymographic gel showing active MMP-2 and-9 bands in 24 h conditioned medium of cultured Egr1-shRNA-expressing SK-HEP-1 cells left untreated or stimulated with tetracycline and/or HGF ( 7D, 7E ). Protein levels of MT1-MMP were studied in tetracyclin and/or HGF-induced SK-HEP-1-Egr1 cells ( 7F ). Densitometric analysis of gelatin zymography and immunoblots showed that HGF induces MT1-MMP expression and MMP-2 and -9 activation, whereas silencing of Egr1 blocks HGF-induced MMPs up-regulation.

Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Stable Transfection, Transfection, Recombinant, Plasmid Preparation, Expressing, shRNA, Migration, Cell Culture, Zymography, Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

doi: 10.1371/journal.pone.0042717

Figure Lengend Snippet: The effect of Egr1 on the cell invasion of HCC cell lines were examined using SNU-449 transiently transfected with pCMV-6-AC-GFP Egr1 overexpression vector ( 8A ). Equal amounts of wild type and Egr1 overexpressing SNU-449 cells were used for migration ( 8B ) and invasion ( 8C ) assay in the presence and absence of HGF. Results are expressed as fold differences of matrigel invaded cells. Zymographic gel showing the active MMP-9 bands in 24 h conditioned medium of cultured Egr1-overexpressing SNU-449 cells ( 8D ). The graph compares the signal intensities obtained from zymography gels.

Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Over Expression, Plasmid Preparation, Migration, Cell Culture, Zymography

Journal: PLoS ONE

Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

doi: 10.1371/journal.pone.0042717

Figure Lengend Snippet: The effects of heparin and HGF on Egr1 expression in Egr1 over-expressing SNU-449 cells was analyzed by immunoblotting ( 9A ). Following serum deprivation, Egr1 overexpressed SN-449 cells were left untreated (0) or treated with HGF in the presence or absence of heparin, then invasion and migration assays were performed using the Roche xCELLigence System ( 9B, 9C ). Results are representative of three or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Migration

Journal: PLoS ONE

Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

doi: 10.1371/journal.pone.0042717

Figure Lengend Snippet: Activation of HGF/c-Met signaling in HCC cells leads to MAPK signaling (i), and up-regulation of Egr1 (ii), which in turn induces MMP-2 and MMP-9 activation and MT1-MMP expression (iii), and increased cell motility and invasion (iv). Heparin treatment results in the inhibition of HGF-induced cellular invasion via repression of HGF-induced c-Met activation, as well as inhibition of MAPK signaling, downregulation of Egr1 expression, and MMP activation (v).

Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Inhibition

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: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

doi: 10.1371/journal.pone.0116825

Figure Lengend Snippet: A. WT1 mRNA (+KTS and –KTS) splice variant PCR products expressed in LβT2 cells. Whole cell RNA was extracted from LβT2 cells, reverse transcribed to cDNA and quantified by real-time PCR, using specific primers to detect +KTS and –KTS splice variants, then displayed on a 1% agarose gel. Bands corresponding to the products for +KTS (301bp) and –KTS (292 bp) WT1 were detected in 4 independent samples of mRNA. B. WT1 and Egr1 protein expression in LβT2 cells. Cell proteins (30μg) were separated on 10% polyacrylamide-SDS gels, then analyzed by immunoblotting with specific antibodies for WT1, Egr1 and β-actin. Specific proteins were detected in the same samples of untreated LβT2 cells. C. Chromatin association of WT1 with the endogenous LHβ promoter in LβT2 cells. The association of WT1 and RNA Polymerase II with the LHβ promoter in untreated LβT2 cells was measured by Chromatin immunoprecipitation assays with antibodies against WT1 and phosphorylated RNApol II, as well as control (no Antibody). LHβ promoter occupancy was measured by quantitative real time PCR using primers specific for the LHβ promoter, and normalized for chromatin input in each sample. In this study, background binding (no Antibody) was set at 100% and association of RNA Polymerase II and WT1 are expressed relative to background values. Association was measured in 3 independent experiments with duplicate samples.

Article Snippet: Membranes were then incubated with a WT1 primary antibody (C-19:SC-192 Santa Cruz Biotechnologies, Santa Cruz CA) overnight (1:500) or Egr1 primary antibody (Cell Signaling Technology) overnight (1:1000) at 4C followed by three 5 min washes with TBST and another incubation with secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit Fab fragment IgG (1:5000;GE HealthCare; Piscataway, New Jersey) for 2h.

Techniques: Variant Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Western Blot, Chromatin Immunoprecipitation, Binding Assay

Journal: PLoS ONE

Article Title: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

doi: 10.1371/journal.pone.0116825

Figure Lengend Snippet: A. WT1 (-KTS) and (+ KTS) mRNA variant levels in LβT2 cells treated with 50nM GnRH for 90min. RNA was extracted and mRNAs levels measured by RT-PCR and normalized against GAPDH mRNA. Values for WT1 mRNAs are shown for both Reverse Transcriptase (RT) and no RT (negative control) conditions. Note that for no RT, the Y axis is interrupted and expanded to show the low values. Data is the mean ± SEM from 5–7 experiments. * = P<0.05, -GnRH vs +GnRH; ** = P<0.001, -GnRH vs +GnRH B. WT1 and Egr1 protein levels in LβT2 cells treated with 50nM GnRH for 0 to 3.5 h. The experiment was performed three times. Upper panel: Cells were lysed after GnRH treatment and proteins (30μg) were separated by 10% SDS-PAGE, then detected with antibodies against WT1 or Egr1. Immunoblotting for β-actin was performed on the same blots as WT1 and Egr1, and used for normalization of these proteins quantified by densitometry analysis. A representative blot is shown. Lower panel: Quantification of protein bands was performed with densitometry, and normalized protein levels are shown from combined experiments. Bands for Egr1 proteins were not detected (ND) in any blot at time zero without GnRH.

Article Snippet: Membranes were then incubated with a WT1 primary antibody (C-19:SC-192 Santa Cruz Biotechnologies, Santa Cruz CA) overnight (1:500) or Egr1 primary antibody (Cell Signaling Technology) overnight (1:1000) at 4C followed by three 5 min washes with TBST and another incubation with secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit Fab fragment IgG (1:5000;GE HealthCare; Piscataway, New Jersey) for 2h.

Techniques: Variant Assay, Reverse Transcription Polymerase Chain Reaction, Negative Control, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

doi: 10.1371/journal.pone.0116825

Figure Lengend Snippet: LβT2 cells were incubated with or without 50 nM GnRH and collected every 10 min for 120 min. ChIP assays were performed using antibody against WT1, Egr1 and phosphorylated pRNA Pol II. LHβ promoter occupancy was measured by quantitative real time PCR using primers for the LHβ promoter. The experiment was performed three times with duplicate samples and replicate PCR measurements in each sample. Data are presented as the mean + SEM and expressed as LHβ promoter occupancy relative to basal (no GnRH at time zero) binding.

Article Snippet: Membranes were then incubated with a WT1 primary antibody (C-19:SC-192 Santa Cruz Biotechnologies, Santa Cruz CA) overnight (1:500) or Egr1 primary antibody (Cell Signaling Technology) overnight (1:1000) at 4C followed by three 5 min washes with TBST and another incubation with secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit Fab fragment IgG (1:5000;GE HealthCare; Piscataway, New Jersey) for 2h.

Techniques: Incubation, Real-time Polymerase Chain Reaction, Binding Assay

Journal: PLoS ONE

Article Title: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

doi: 10.1371/journal.pone.0116825

Figure Lengend Snippet: LβT2 cells were transfected with siRNA against WT1 and a non-targeting siRNA as a control. After 72 h of incubation, cells were treated with or without GnRH for 1.5 h, followed by cell lysate collection, RNA extraction and western blot analysis (30 μg protein) on 10% PAGE-SDS gels. WT1 protein was detected by immunoblotting with specific antibodies for WT1, and normalized for β-actin on the same blot. A. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where the WT1 (-KTS) variant mRNA was reduced. B. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where both the WT1 (-KTS) and WT1 (+KTS) variant mRNAs were reduced. For each condition, the experiment was performed twice with 5 replicates. LHβ and Egr1 primary transcript mRNAs were normalized for GAPDH mRNA in the same sample. Control samples contained non-targeting siRNA. * p<.05 -GnRH vs +GnRH, in either Control siRNA or WT1 siRNA treatments. Values are mean ± SEM for 5 replicates. # = p<.05 -GnRH Control siRNA vs –GnRH WT1 siRNA, or p<.05+GnRH Control siRNA vs +GnRH WT1 siRNA.

Article Snippet: Membranes were then incubated with a WT1 primary antibody (C-19:SC-192 Santa Cruz Biotechnologies, Santa Cruz CA) overnight (1:500) or Egr1 primary antibody (Cell Signaling Technology) overnight (1:1000) at 4C followed by three 5 min washes with TBST and another incubation with secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit Fab fragment IgG (1:5000;GE HealthCare; Piscataway, New Jersey) for 2h.

Techniques: Transfection, Incubation, RNA Extraction, Western Blot, Expressing, Variant Assay

Journal: PLoS ONE

Article Title: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

doi: 10.1371/journal.pone.0116825

Figure Lengend Snippet: WT1 (-KTS) may associate directly with the 3’Egr1 site of the LHβ promoter to stimulate basal promoter activity, but in the presence of GnRH stimulates Egr1 expression. Egr1 then binds to both the 3’- and 5’-Egr1 sites on the promoter and further increases LHβ transcription. WT1 (+KTS) may associate with the 3’-Egr1 site, but also requires the SF1 site for biological activity. Decreased expression of WT1 (+KTS) would stimulate basal LHβ expression as the suppressor is reduced. The isoforms likely compete for association to the LHβ promoter at the same sites.

Article Snippet: Membranes were then incubated with a WT1 primary antibody (C-19:SC-192 Santa Cruz Biotechnologies, Santa Cruz CA) overnight (1:500) or Egr1 primary antibody (Cell Signaling Technology) overnight (1:1000) at 4C followed by three 5 min washes with TBST and another incubation with secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit Fab fragment IgG (1:5000;GE HealthCare; Piscataway, New Jersey) for 2h.

Techniques: Activity Assay, Expressing

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: International Journal of Medical Sciences

Article Title: Expression profiling of lncRNAs and mRNAs in placental site trophoblastic tumor (PSTT) by microarray

doi: 10.7150/ijms.65002

Figure Lengend Snippet: The sequence of the primers for lncRNAs and mRNAs

Article Snippet: EGR1 antibody (4154S) was purchased from Cell Signaling Technology (San Diego, CA, USA).

Techniques: Sequencing

Journal: International Journal of Medical Sciences

Article Title: Expression profiling of lncRNAs and mRNAs in placental site trophoblastic tumor (PSTT) by microarray

doi: 10.7150/ijms.65002

Figure Lengend Snippet: Validation of the microarray results of mRNAs and lncRNAs by RT-qPCR and immunohistochemistry . RT-qPCR was performed to test the differentially expressed mRNAs (A) and lncRNAs (C) (n=4 for PSTT group and control group, respectively); the fold change of each mRNA (B) and lncRNA (D) between PSTT tissues and control group was determined microarray and RT-qPCR. The expression of PLAC8 and EGR1 was obviously higher than normal villi, while ADAMTS6 expression is much lower in PSTT compared to normal villi (F). ns, No significance; *, P < 0.05; **, P < 0.01, P < 0.001, Student t -test. Scale bar = 100μm.

Article Snippet: EGR1 antibody (4154S) was purchased from Cell Signaling Technology (San Diego, CA, USA).

Techniques: Microarray, Quantitative RT-PCR, Immunohistochemistry, Expressing

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: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: Imaging analysis. (A) Confocal images of EGR1 and fibrillarin (upper row) or EGR1 and B23 (lower row) in the nucleolus of HeLa cells. The images were obtained with a Leica SP2 and analyzed under HCX PL APO CS 63x. (B) Immunogold electron microscopy (EM) labeling of EGR1 in the fibrillar center of the nucleolus. Ultrathin sections of HeLa cells were embedded in Lowicryl K4M. (FC, Fibrillar center; DFC, Dense Fibrillar Component; GC, Granular Component. Arrow: labelling in the CF; arrowheads: labelling in the nucleoplasm).

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit polyclonal against N-terminal of EGR1 (4153, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal antibody anti-fibrillarin (ab4566, Abcam, Cambridge, MA, USA), mouse monoclonal antibody anti-B23 (ab10530, Abcam) and mouse monoclonal anti-UBF antibody (sc-13125, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Imaging, Electron Microscopy, Labeling

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: Biochemical evidences. (A) Detection by immunoblotting of EGR1 in nuclei, nuclear and nucleolar extracts of HeLa grown at 0.2% or 10% FBS. The sumoylated form of EGR1 present within the nucleolar extracts is detected by an anti-sumo1 antibody (Sigma-Aldrich). The band signals were quantitated for comparison between extracts of cells grown at 0.2% and 10% FBS. Signals were normalized to the loading control. Beta-tubulin and fibrillarin are shown as loading control. (B) Immunofluorescence of EGR1 after treatment with actinomycin D (0.04 µg/ml) for 1h at 37°C. HeLa cells were treated and stained for EGR1 and fibrillarin by immunofluorescence. The images were taken by under a 40X objective with a LEICA DM4000B. The pictures at the right show the merging of the two fluorescing proteins. (C) Nuclei of HeLa cells were extracted and immunoblotted to quantitate the expression of EGR1 following Actinomycin D treatment. Both immunofluorescence and western blotting show that the endogenous levels of EGR1 are not significantly affected by the treatment. Representative results of at least three separate experiments are shown. Comparison tests were assessed by one way ANOVA, and significances are shown where applicable. Asterisk (*) represent p≤0.05 when compared to relative controls.

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit polyclonal against N-terminal of EGR1 (4153, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal antibody anti-fibrillarin (ab4566, Abcam, Cambridge, MA, USA), mouse monoclonal antibody anti-B23 (ab10530, Abcam) and mouse monoclonal anti-UBF antibody (sc-13125, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Western Blot, Immunofluorescence, Staining, Expressing

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: Confocal images of HeLa cells transfected with (A) the full length EGR1 (1–543 AA), (B) the N-terminal (ΔC-EGR1) (1–314 AA), (C) the C-terminal EGR1 (ΔN-EGR1) (315–543 AA). Each construct was fused to the GFP. (D) Empty pEGFP vector. Full length EGR1, N-terminal EGR1, C-terminal EGR1 and stained with an antibody to fibrillarin.

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit polyclonal against N-terminal of EGR1 (4153, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal antibody anti-fibrillarin (ab4566, Abcam, Cambridge, MA, USA), mouse monoclonal antibody anti-B23 (ab10530, Abcam) and mouse monoclonal anti-UBF antibody (sc-13125, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Transfection, Construct, Plasmid Preparation, Staining

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: (A) The synthesis of 47S rRNA is strongly upregulated following inhibition of EGR1. 47S synthesis in Hela cells grown in 0.2% FBS is significantly increased following endogenous EGR1 silencing with either 10 nM or 15 nM specific siRNA (middle graph). 47S synthesis is not affected in cells treated with a scrambled sequence compared to the untreated cells taken as control. The levels of expression of EGR1 and p300 following the siRNA treatment are shown in the left and right graphs, respectively. Both levels are significantly diminished after EGR1 silencing. (B) 47S synthesis in HeLa cells grown in 0.2% or 10% FBS is significantly depressed after transfection of full length EGR1. The levels of expression of EGR1 and p300 are shown in the left and right graphs, respectively. As expected, both levels are significantly upregulated after EGR1 transfection compared to control cells. Representative results of at least three separate experiments are shown. Comparison tests were performed by one way ANOVA, and significant results are highlighted with asterisks (* p≤0.05, ** p≤0.01 in comparison with relative controls).

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit polyclonal against N-terminal of EGR1 (4153, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal antibody anti-fibrillarin (ab4566, Abcam, Cambridge, MA, USA), mouse monoclonal antibody anti-B23 (ab10530, Abcam) and mouse monoclonal anti-UBF antibody (sc-13125, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Inhibition, Sequencing, Expressing, Transfection

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: (A) The synthesis of 47S rRNA in NIH 3T3 (ARF−/−) cells grown in 0.2% FBS (right graph) is not affected when the expression of endogenous EGR1 is silenced with 10 nM of specific siRNA (left graph). (B) Viceversa, the synthesis of 47S rRNA (left graph) is greatly reduced when both EGR1 (middle graph) and p19ARF genes (right graph) are transfected and expressed in NIH 3T3 following transfection with plasmid expression vectors. Transfection with pEGFP is shown as control. Comparison tests were assessed by one way ANOVA, and significant differences are highlighted with asterisks (* p<0.05; ** p<0.01).

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit polyclonal against N-terminal of EGR1 (4153, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal antibody anti-fibrillarin (ab4566, Abcam, Cambridge, MA, USA), mouse monoclonal antibody anti-B23 (ab10530, Abcam) and mouse monoclonal anti-UBF antibody (sc-13125, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: The Transcription Factor EGR1 Localizes to the Nucleolus and Is Linked to Suppression of Ribosomal Precursor Synthesis

doi: 10.1371/journal.pone.0096037

Figure Lengend Snippet: (A) Confocal analysis of Hela cells transfected with the C-terminal EGR1 (ΔN 1–314) shows the colocalization of EGR1 fragment with UBF. (B) Extracts (150 µg) of HeLa cells transfected with full length EGR1-GFP are immunoprecipitated with an antibody to UBF. (C) Chromatin precipitation assay. DNA fragments of ribosomal RNA promoter are immunoprecipitated with an antibody to EGR1 from extracts of HeLa cells transfected with full length EGR1. A six fold enrichment in ribosomal RNA promoter fragments was obtained from extracts of transfected cells compared to mock extracts. Representative results of at least three separate experiments are shown. Comparison tests were performed as above described (** p≤0.01).

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit polyclonal against N-terminal of EGR1 (4153, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal antibody anti-fibrillarin (ab4566, Abcam, Cambridge, MA, USA), mouse monoclonal antibody anti-B23 (ab10530, Abcam) and mouse monoclonal anti-UBF antibody (sc-13125, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Transfection, Immunoprecipitation