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