Journal: Science Advances

Article Title: Influenza virus replication in cardiomyocytes drives heart dysfunction and fibrosis

doi: 10.1126/sciadv.abm5371

Figure Lengend Snippet: WT and IFITM3 KO mice were intranasally infected with PR8-miR133b/206 or PR8-miRctrl (50 TCID 50 ). ( A ) Hearts were collected on day 10 after infection, and sections were stained with Masson’s trichrome stain, in which blue staining is indicative of fibrotic collagen deposition. Histological processing and image acquisition were performed by the OSU Comparative Pathology and Mouse Phenotyping Core Facility on heart tissue samples provided by A.D.K. A representative heart section is shown for each genotype-virus combination. Boxed areas are regions magnified in the far-right images. Scale bars, 1 mm and 200 μm for the left and right images, respectively. ( B ) Percent fibrosis was calculated by quantifying ratio of blue pixel intensity to total pixel intensity for each heart section. Each point represents a heart from an individual mouse, and bars represent mean values. Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s post hoc test. * P < 0.05. ( C ) Serum from IFITM3 KO mice was collected before infection and at days 5 and 10 after infection with PR8-miRctrl or PR8-miR133b/206 for ELISA quantification of creatine kinase. Data points represent individual mice, and bars represent mean values. Error bars depict SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s post hoc test. * P < 0.05.

Article Snippet: Creatine kinase quantification was performed using the Mouse Creatine Kinase MB ELISA Kit (Novus Biologicals).

Techniques: Infection, Staining, Virus, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: RAGE activation in macrophages and development of experimental diabetic polyneuropathy

doi: 10.1172/jci.insight.160555

Figure Lengend Snippet: ( A ) Kymographs of LysoTracker-labeled organelles in axons from dorsal root ganglia (DRG) neurons with 1.0 U/mL insulin. The horizontal and vertical arrows indicate retrograde direction and recording time (4 minutes), respectively. ( B – E ) The percentage of organelles in 100 μm axon segments that moved anterogradely ( B ), retrogradely ( C ), bidirectionally ( D ), or were stationary ( E ). n = 18–21 axons from 3 independent experiments. ( F ) The velocity of retrograde movements (RV) in 100 μm axon segments. The data consisted of 200–300 movements. ( G ) Kymographs in axons from DRG neurons treated with vehicle and insulin receptor antagonist (BMS-754807, 300 or 500 nmol/L). The stimulation time was 60 minutes. The horizontal and vertical arrows indicate retrograde direction and recording time (4 minutes), respectively. ( H – K ) The percentage of organelles in 100 μm axon segments that moved anterogradely ( H ), retrogradely ( I ), or bidirectionally ( J ), or were stationary ( K ). n = 18–21 axons from 3 independent experiments. ( L ) RV in 100 μm axon segments in each treatment condition. ( M ) Kymographs in axons from DRG neurons treated with vehicle, TNF-α, and TNF-α + JNK inhibitor (SP600125). The stimulation time was 20 minutes. The vertical arrow indicates recording time (4 minutes). ( N – Q ) The percentage of organelles in 100 μm axon segments that moved anterogradely ( N ), retrogradely ( O ), bidirectionally ( P ), or were stationary ( Q ). n = 18–21 axons from 3 independent experiments. ( R ) RV in 100 μm axon segments. The data consisted of 200–300 movements. The data are presented as the mean ± SD. Because the experiments of G – L and M – R were performed contemporaneously, statistical analysis was done using same vehicle control. Statistical analysis was performed by Student’s 2-tailed unpaired t test for B – F and by 1-way ANOVA with Tukey’s multiple-comparison test for H – L and N – R . ** P < 0.01, *** P < 0.001.

Article Snippet: In the experiments with an insulin/IGF-1 receptor antagonist shown in , or with TNF-α with or without a JNK inhibitor shown in , neurons were maintained in DMEM/F12 with 2% B27 and were pretreated with PBS containing 0.1% BSA (vehicle), 300 nmol/L, or 500 nmol/L BMS-754807 (MedChemExpress); 20 ng/mL TNF-α (410-MT, R&D Systems); or 20 ng/mL TNF-α + 50 nmol/L JNK inhibitor (SP600125) (1496, TOCRIS) as indicated in the text.

Techniques: Labeling, Control, Comparison

Journal: BMC Cell Biology

Article Title: Peptide aptamers as new tools to modulate clathrin-mediated internalisation — inhibition of MT1-MMP internalisation

doi: 10.1186/1471-2121-11-58

Figure Lengend Snippet: Isolation and validation of PAs interacting with MT1-MMP ICD . (A) Schematic representation of the TrxA scaffold and the PAs swiggle, 76, and s14. The sequences of the peptide inserted in TrxA (swiggle, s14) or fused to the N-terminal region of TrxA (76) are detailed. The point mutation in the sequence of s14 is depicted in bold and the amino acids in lower cases in PA 76 represent linkers between the multiple peptides. * denotes the stop codon. (B) Swiggle interacts with the MT1-MMP ICD in a yeast-two hybrid interaction assay. EGY48 cells expressing LexA-DBD, LexA-MT1, LexA-MT2, LexA-MT3, LexA-MT5 or LexA-Cdk4 were mated with EGY42 cells expressing AD-TrxA, AD-swiggle, AD-s14, AD-76 or AD-CyclinD1 and plated onto selective media.

Article Snippet: Oligonucleotides coding for a PGGG linker followed by MT1-MMP, MT2-MMP, MT3-MMP, or MT5-MMP intracellular domain (ICD) were annealed and cloned downstream of the LexA DNA binding domain (DBD) in pEG202 (Origene, Rockville, USA) to generate LexA-MT1, -MT2, -MT3 and -MT5.

Techniques: Isolation, Mutagenesis, Sequencing, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

doi: 10.1074/jbc.RA120.012635

Figure Lengend Snippet: Malaysia/B HA is proteolytically activated by murine membrane-anchored proteases TMPRSS4, TMPRSS13, hepsin, and prostasin, but not matriptase. A, examination of HA cleavage. HEK293 cells were co-transfected with plasmids encoding Malaysia/B HA and either TMPRSS2, TMPRSS4, TMPRSS13, prostasin, hepsin, or matriptase. Co-transfection of HA-expressing plasmid and empty vector (w/o) and transfection of empty vector only (ev) were used as control. Cell lysates were subjected to SDS-PAGE and Western blot analysis at 48 h post-transfection using HA-specific antibodies. β-Actin served as loading control. B, proteolytic activation and multicycle replication analysis. MDCK cells were transfected with protease-encoding plasmids for 24 h and then infected with Malaysia/B at a MOI 0.01. At 24 h p.i., cells were fixed and immunostained against the viral NP to examine virus spread. Transfection of cells with empty vector (ev) was used as negative control. Data are representatives of at least three independent experiments. C, cleavage of H9 by matriptase. HEK293 cells were co-transfected with plasmids encoding H9 of A/quail/Shantou/782/00 (H9N2) and either TMPRSS2 or matriptase. Co-transfection of H9-expressing plasmid and empty vector (w/o) or transfection of empty vector only (ev) were used as control. Cell lysates were analyzed by SDS-PAGE and Western blotting at 48 h post-transfection using H9-specific antibodies. β-Actin served as loading control. D, MDCK cells were transfected with protease-encoding plasmids for 24 h and then infected with H9N2 at an MOI of 0.03 for 24 h to allow multicycle replication. Cells were fixed and immunostained against NP. Transfection of cells with empty vector (ev) was used as negative control. Data are representative of three independent experiments.

Article Snippet: The cDNA of the HA gene of B/Malaysia/2506/2004 was cloned from viral RNA by RT-PCR using HA-specific primers and subsequently subcloned into pCAGGS expression plasmid using EcoRI and NotI restriction sites. pCMV6-Entry expression plasmids encoding murine proteases with a C-terminal Myc-DDK tag were obtained from OriGene Technologies: CFB (MR210521), HGFA (MR216475), hepsin (MR219750), LTF (MR210170), tPA (MR208868), uPA (MR225747), tryptase ϵ (MR204321), NSP4 (MR217041), prostasin (MR223227), matriptase (MR222240), TMPRSS4 (MR206946), and TMPRSS13 (MR220731). pCAGGS plasmids encoding human or murine TMPRSS2 with C-terminal FLAG epitope have been described previously ( 6 , 15 ). pCAGGS encoding human prostasin was described previously ( 31 ). pcDNA6.2/C-emGFP-HPN encoding human hepsin (Human ORFeome Collaboration, Clone ID: 100004749) was generously provided by the Juha Klefström laboratory (University of Helsinki).

Techniques: Transfection, Cotransfection, Expressing, Plasmid Preparation, SDS Page, Western Blot, Activation Assay, Infection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

doi: 10.1074/jbc.RA120.012635

Figure Lengend Snippet: Malaysia/B HA is not activated by murine tPA, uPA, LTF, NSP4, CFB, tryptase ϵ, and HGFA. A, examination of HA cleavage by soluble proteases present in cell supernatants. HEK293 cells with transient expression of Malaysia/B HA were incubated with cleared protease containing HEK293 cell supernatants as described under “Experimental procedures” (left) or recombinant rKLK8 (right). Treatment of HA-expressing cells with buffer (w/o) or trypsin was used as control. Cell lysates were analyzed for HA cleavage by immunoblotting. β-Actin was used as loading control. B, MDCK cells with transient protease expression were infected with Malaysia/B at a low MOI of 0.01 and incubated for 24 h to allow multicycle viral replication. Cells transfected with empty vector (ev) or murine TMPRSS2-expressing plasmid were used as control. Virus spread was visualized by immunostaining of infected cells against NP. C, expression analysis of tryptase ϵ_DDDDK mutant in HEK293 cells with or without enterokinase treatment. Supernatants of cells transfected with empty vector (ev) or tryptase ϵ_DDDDK-encoding plasmid were concentrated (5×) at 48 h post-transfection and analyzed by SDS-PAGE and Western blotting using tryptase ϵ–specific antibodies. Zymogen and mature form are indicated by filled and open arrowheads, respectively. D, examination of HA cleavage by tryptase ϵ. HEK293 cells expressing Malaysia/B HA were incubated with tryptase ϵ_DDDDK mutant–containing cell supernatants treated with or without enterokinase (10 IU). Treatment of HA-expressing cells with trypsin was used as control. Cell lysates were analyzed for HA cleavage. E, expression analysis of HGFA in HEK293 supernatants with and without matriptase treatment. At 48 h post-transfection with empty vector (ev) or HGFA-encoding plasmid cell supernatants were concentrated (5×), treated with or without matriptase (5.0 μg/ml) for 1 h at 37 °C, and analyzed by immunoblotting using a FLAG-specific antibody. Zymogen and mature form are indicated by filled and open arrowheads, respectively. F, examination of HA cleavage by HGFA. HEK293 cells co-transfected with plasmids encoding Malaysia/B HA and either empty vector (w/o) or HGFA-encoding plasmid were incubated with exogenous matriptase or trypsin (0.5 μg/ml each) or remained untreated for 24 h. Cell lysates were analyzed for HA cleavage by Western blotting.

Article Snippet: The cDNA of the HA gene of B/Malaysia/2506/2004 was cloned from viral RNA by RT-PCR using HA-specific primers and subsequently subcloned into pCAGGS expression plasmid using EcoRI and NotI restriction sites. pCMV6-Entry expression plasmids encoding murine proteases with a C-terminal Myc-DDK tag were obtained from OriGene Technologies: CFB (MR210521), HGFA (MR216475), hepsin (MR219750), LTF (MR210170), tPA (MR208868), uPA (MR225747), tryptase ϵ (MR204321), NSP4 (MR217041), prostasin (MR223227), matriptase (MR222240), TMPRSS4 (MR206946), and TMPRSS13 (MR220731). pCAGGS plasmids encoding human or murine TMPRSS2 with C-terminal FLAG epitope have been described previously ( 6 , 15 ). pCAGGS encoding human prostasin was described previously ( 31 ). pcDNA6.2/C-emGFP-HPN encoding human hepsin (Human ORFeome Collaboration, Clone ID: 100004749) was generously provided by the Juha Klefström laboratory (University of Helsinki).

Techniques: Expressing, Incubation, Recombinant, Western Blot, Infection, Transfection, Plasmid Preparation, Immunostaining, Mutagenesis, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

doi: 10.1074/jbc.RA120.012635

Figure Lengend Snippet: Cleavage of IBV HA and H3 by protease candidates and confirmed protease activity

Article Snippet: The cDNA of the HA gene of B/Malaysia/2506/2004 was cloned from viral RNA by RT-PCR using HA-specific primers and subsequently subcloned into pCAGGS expression plasmid using EcoRI and NotI restriction sites. pCMV6-Entry expression plasmids encoding murine proteases with a C-terminal Myc-DDK tag were obtained from OriGene Technologies: CFB (MR210521), HGFA (MR216475), hepsin (MR219750), LTF (MR210170), tPA (MR208868), uPA (MR225747), tryptase ϵ (MR204321), NSP4 (MR217041), prostasin (MR223227), matriptase (MR222240), TMPRSS4 (MR206946), and TMPRSS13 (MR220731). pCAGGS plasmids encoding human or murine TMPRSS2 with C-terminal FLAG epitope have been described previously ( 6 , 15 ). pCAGGS encoding human prostasin was described previously ( 31 ). pcDNA6.2/C-emGFP-HPN encoding human hepsin (Human ORFeome Collaboration, Clone ID: 100004749) was generously provided by the Juha Klefström laboratory (University of Helsinki).

Techniques: Activity Assay

Journal: Journal of Clinical Medicine

Article Title: Paradoxical Changes: EMMPRIN Tissue and Plasma Levels in Marfan Syndrome-Related Thoracic Aortic Aneurysms

doi: 10.3390/jcm13061548

Figure Lengend Snippet: MT1-MMP is elevated in MFS TAA Tissue. ( A ) Representative total protein and immunoblot for the full-length, active form of MT1-MMP (64 kDa) abundance in control and MFS TAA tissue. ( B ) Fold change in MT1-MMP abundance in MFS TAA tissue compared to control tissues (dashed line). The mean is depicted as a solid red bar ± the standard error of the mean. The median is depicted as an X. All data points are shown. * p < 0.05 vs. control (Student’s t -test).

Article Snippet: In Vitro Transfection: Aortic fibroblasts were transfected for 18 h using jetPRIME transfection reagent (114-15, Polypolus-transfection Illkirch-Graffenstaden, France) according to the DNA transfection protocol with either 10 µg MT1-MMP over expression vector (MC219253, OriGene, Rockville, MD, USA) or the transfection reagent alone.

Techniques: Western Blot, Control

Journal: Journal of Clinical Medicine

Article Title: Paradoxical Changes: EMMPRIN Tissue and Plasma Levels in Marfan Syndrome-Related Thoracic Aortic Aneurysms

doi: 10.3390/jcm13061548

Figure Lengend Snippet: Effects of MT1-MMP abundance and localization on secreted EMMPRIN levels in conditioned culture media. ( A ) Concentration of EMMPRIN in conditioned culture media from aortic fibroblasts transfected with an MT1-MMP over expression vector compared to the control. The mean is depicted as a solid bar ± the standard error of the mean. The median is depicted as an X. All data points are shown. * p < 0.05 vs. control (Student’s t -test). ( B ) Concentration of EMMPRIN in conditioned culture media from aortic fibroblasts treated with PMA or Röttlerin compared to the control. The mean is depicted as a solid bar ± the standard error of the mean. The median is depicted as an X. All data points are shown. * p < 0.05 vs. control (ANOVA, post hoc test: Holm–Sidak); # p < 0.05 vs. PMA (ANOVA, post hoc test: Holm–Sidak).

Article Snippet: In Vitro Transfection: Aortic fibroblasts were transfected for 18 h using jetPRIME transfection reagent (114-15, Polypolus-transfection Illkirch-Graffenstaden, France) according to the DNA transfection protocol with either 10 µg MT1-MMP over expression vector (MC219253, OriGene, Rockville, MD, USA) or the transfection reagent alone.

Techniques: Concentration Assay, Transfection, Over Expression, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) Relative mRNA levels of TCF-4 and MMP-15 in SAEC, H460, A549 and LLC cells (n = 5, **P < 0.01). ( b ) Relative mRNA levels of TCF-4 and MMP-15 in the lung tissues or LLC-tumor tissues. The 8-week old female C57BL/6 mice were intravenously injected with LLC cells (5 × 10 5 cells in 100 μl PBS). Two weeks later, the inoculated LLC-tumors or adjacent lung tissues were collected for mRNA assays (n = 5, **P < 0.01). ( c , d ) Relative mRNA levels of TCF-4 ( c ) and MMP-15 ( d ) in the lung carcinoma or adjacent normal tissues from human subjects (n = 10, **P < 0.01). The experiment in ( a , b ) was performed in triplicate. The experiment in ( c , d ) was repeated twice.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Injection

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a , b ) The mRNA ( a ) and protein ( b ) levels of TCF-4 and MMP-15 in SAEC cells which were transfected with PCMV (0.4 μg/ml) or PCMV-TCF-4 (0.4 μg/ml) for 24 h (n = 5, **P < 0.01). ( c , d ) The mRNA ( c ) and protein ( d ) levels of TCF-4 and MMP-15 in LLC cells which were transfected with a scramble siRNA (si-NC, 20 nmol/ml) or the mouse TCF-4 specific siRNAs (si-TCF4-1 or si-TCF4-2, 20 nmol/ml) for 36 hours (n = 3, *P < 0.05). The experiment from ( a – d ) was repeated in triplicate and the representative results were displayed.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Transfection

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus the reporter gene (m-P1) (0.4 μg/ml) harboring the mouse MMP-15 promoter sequences (-3000/-1) for 36 hours (n = 5, **P < 0.01). ( b ) Relative luciferase activity of SAEC cells transfected with si-NC (20 nmol/ml) or a siRNA for human TCF-4 (si-TCF4, 20 nmol/ml) plus the reporter gene (h-P1) (0.4 μg/ml) harboring the human MMP-15 promoter sequences (-3000/-1) for 36 hours (n = 5, **P < 0.01). ( c ) A schematic depiction of different mouse MMP-15 promoter regions which were cloned into the pGL4-basic plasmid. The constructs were designated as m-P1~m-P3 as indicated. ( d ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus the reporter genes m-P1~m-P3 (0.4 μg/ml) for 36 hours (n = 5, **P < 0.01). The tests in ( a , b , d ) were repeated in triplicate.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Luciferase, Activity Assay, Transfection, Clone Assay, Plasmid Preparation, Construct

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) The conserved promoter region and NF-κB binding sites between human and mouse MMP-15 gene. The human MMP-15 promoter sequences (-3000/-1) were aligned with the mouse MMP-15 promoter sequences (-3000/-1). Two conserved NF-κB binding elements in the mouse MMP-15 promoter (-2958/-2949, -2833/-2824) and human MMP-15 promoter (-2456/-2447, -2348/-2339) were mutated and constructed as reporter genes m-MUT1, m-MUT2, h-MUT1 and h-MUT2, respectively as indicated. ( b ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus the reporter genes m-P1, m-MUT1 and m-MUT2 (0.4 μg/ml) for 36 hours, respectively (n = 5, **P < 0.01). ( c ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus the reporter genes h-P1, h-MUT1 and h-MUT2 (0.4 μg/ml) for 36 hours, respectively (n=5, **P < 0.01). The experiment in ( b , c ) was repeated for 3 times.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Binding Assay, Construct, Luciferase, Activity Assay, Transfection

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) TCF-4 potentiates the binding between NF-κB p65 and the MMP-15 promoter. The LLC cells were transfected with si-NC (20 nmol/ml), si-TCF4-1 (20 nmol/ml) or si-TCF4-2 (20 nmol/ml) for 36 hours, and then harvested for ChIP assay. The cell lysates were immunoprecipitated by p65 antibody or rabbit IgG as control. ( b ) TCF-4 interacts with NF-κB p65 which binds to the promoter of MMP-15. The LLC cells were transfected with si-NC (20 nmol/ml), si-TCF4-1 (20 nmol/ml) or si-TCF4-2 (20 nmol/ml) for 36 hours, and then harvested for ChIP assay. The cell lysates were immunoprecipitated by TCF-4 antibody or rabbit IgG as control. ( c ) Protein-protein interaction between TCF-4 and NF-κB p65 in LLC cells. The LLC cells were transfected with si-NC (20 nmol/ml), si-TCF4-1 (20 nmol/ml) or si-TCF4-2 (20 nmol/ml) for 36 hours, and then harvested for immunoprecipitation (IP) and Western blotting assay of p65 protein. The cell lysates were immunoprecipitated by TCF-4 antibody or rabbit IgG as control. ( d ) Protein-protein interaction between TCF-4 and NF-κB p65 in SAEC cells. The SAEC cells were transfected with PCMV (0.4 μg/ml) or PCMV-TCF4 (0.4 μg/ml) for 36 hours, and then collected for IP and Western blotting assay of p65. The cell lysates were immunoprecipitated by TCF-4 antibody or rabbit IgG as control. Experiment from ( a – d ) was repeated for 3 times, and the representative results were displayed.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) The mRNA levels of MMP-15, TCF-4 and p65 in the LLC cells transfected with PCMV (0.4 μg/ml) or PCMV-TCF-4 (0.4 μg/ml) plus si-NC (20 nmol/ml) or a siRNA for mouse NF-κB p65 (si-p65, 20 nmol/ml) for 36 hours (n = 5, **P < 0.01). ( b ) The mRNA levels of MMP-15, TCF-4 and p65 in the LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus pCDNA3.1 (0.4 μg/ml) or pCDNA-p65 (0.4 μg/ml) for 36 hours (n = 5, **P < 0.01). ( c ) Immunoblotting assay of TCF-4 and p65 in the cytosol or nucleus of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) for 36 hours. ( d ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus a reporter construct (0.4 μg/ml) containing the promoter of CCL20 for 36 hours (n = 4, **P < 0.01). The tests from ( a – d ) were repeated for 3 times, and the representative results were displayed.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Transfection, Western Blot, Luciferase, Activity Assay, Construct

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) TCF-4 promotes LLC cell migration via MMP-15. The scratch tests were performed on the LLC cells which were transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus PCMV (0.4 μg/ml) or PCMV-MMP-15 (0.4 μg/ml). The representative pictures were taken immediately after scratch (0 hours) or after a subsequent 18 hours (18 h). ( b ) Relative migration rate of the cells described in ( a ) (n = 5, **P < 0.01). ( c ) TCF-4 promotes MMP-15-dependent LLC cell migration. Transwell assays were carried out on the cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus PCMV (0.4 μg/ml) or PCMV-MMP-15 (0.4 μg/ml). ( d ) Relative migrated cells described in ( c ) were counted (n = 5, **P < 0.01). The tests in ( a , c ) were repeated for 3 times and the representative images were displayed.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Migration, Transfection

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) Representative images of LLC-tumors in the lung tissues. The LLC cells were transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus PCMV (0.4 μg/ml) or PCMV-MMP-15 (0.4 μg/ml) for 24 hours. Then, the 8-week old female C57BL/6 mice were intravenously injected with those different LLC cells (5 × 10 5 cells in 100 μl PBS) as indicated. Two weeks later, the lung tissues were collected for pathological observation with H&E staining. ( b ) Relative tumor lesions displayed in ( a ) were calculated (n = 6, **P < 0.01). ( c ) Survival time of different LLC cells-inoculated mice. The 8-week old female C57BL/6 mice were intravenously injected with different LLC cells (5 × 10 6 cells in 100 μl PBS) as indicated. The survival time after tumor inoculation was recorded (n = 10, **P < 0.01). The experiment in ( a , c ) was repeated twice and the representative results were displayed.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Transfection, Injection, Staining