Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: Expression levels of MFAP4 in public datasets and human cirrhotic liver tissues. ( A ) Expression of MFAP4 in the GEO dataset ( GSE14323 ) and its correlation with COL1A1, ACTA2, and TIMP1 expression. ( B ) Expression of MFAP4 in the GEO dataset ( GSE84044 ), its association with liver fibrosis grades, and its correlation with COL1A1, ACTA2, and TIMP1 expression. ( C ) Representative images of immunohistochemical staining for MFAP4, COL1, and α-SMA, along with Sirius Red staining, in healthy human liver tissues and cirrhotic human liver tissues. Quantification of the positively stained area were shown at right . ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. Scale bar = 50 μm.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: Expression levels of MFAP4 human cirrhotic liver tissues. Immunohistochemical staining for MFAP4, Col1, and α-SMA, along with Sirius Red staining, in healthy human liver tissues and cirrhotic human liver tissues. Scale bar = 50 μm.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: Successful establishment of 2 liver fibrosis mouse models, and elevated expression of MFAP4 in fibrotic liver tissues and activated HSCs. ( A ) Representative images of HE staining, Sirius Red staining, and immunohistochemical staining of Col1, α-SMA, and MFAP4 in liver tissues after 6 weeks of CCl 4 induction and ( B ) quantification of the positive staining areas (n = 6 mice/treatment groups). ( C ) Representative images of liver histology after 12 weeks of TAA induction, including HE staining, Sirius Red staining, and immunohistochemistry for Col1, α-SMA, and MFAP4 and ( D ) quantification of the positive staining areas (n = 6 mice/treatment groups). ( E ) qPCR analysis of the expression levels of Col1a1, Acta2, TIMP1, and MFAP4 in total mRNA extracted from CCl 4 -induced fibrotic liver tissues (n = 6 mice/treatment groups). ( F ) qPCR analysis of the expression levels of Col1a1, Acta2, TIMP1, and MFAP4 in total mRNA extracted from TAA-induced fibrotic liver tissues (n = 6 mice/treatment groups). ( I ) qPCR analysis of the mRNA levels of Col1a1, Acta2, TIMP1, and MFAP4 in LX-2 cells after 24 hours of incubation with 10 ng/mL TGF-β1. Western blot analysis of the expression levels of Col1, α-SMA, TIMP1, and MFAP4 in CCl 4 ( G ) or TAA ( H ) induced liver fibrosis models (n = 6 mice/treatment groups). ( J ) Western blot analysis of the expression levels of Col1, α-SMA, TIMP1, and MFAP4 in LX-2 cells after 24 hours of incubation with 10 ng/mL TGF-β1. ( K ) Enzyme-linked immunosorbent assay analysis of MFAP4 levels in the culture medium of LX-2 cells after 24-hour incubation with 10 ng/mL TGF-β1. ∗∗ P < .01, ∗∗∗ P < .001. Scale bars: 50 μm in ×100 magnification, 25 μm in ×200 magnification.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Expressing, Staining, Immunohistochemical staining, Immunohistochemistry, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: Single-cell transcriptomic profiling of CCl 4 -induced fibrosis. ( A-C ) UMAP plots showing intrahepatic cell clusters and experimental groups, with FeaturePlot displaying Mfap4 expression in hepatic cells. ( D-F ) UMAP plots of HSC subclusters and experimental groups, with FeaturePlot showing Mfap4 expression in HSCs.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Single Cell, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: MFAP4 deficiency alleviates liver fibrosis induced by CCl 4 and TAA. ( A ) Serum levels of alanine aminotransferase (ALT) (U/L) and aspartate aminotransferase (AST) (U/L) in WT+Oil, WT+CCl 4 , and KO+CCl 4 mice groups (n = 5 mice/treatment groups). ( B ) Serum levels of ALT (U/L) and AST (U/L) in WT+TAA, WT+TAA, and KO+TAA mice groups (n = 5 mice/treatment groups). ( C ) Representative images of HE staining, Sirius Red staining, and immunohistochemical staining for Col1, α-SMA, and MFAP4 in liver tissues from WT+Oil, KO+Oil, WT+CCl 4 , and KO+CCl 4 groups after 6 weeks of treatment. Quantification of the positive staining areas is shown below (n = 6 mice/treatment groups). ( D ) Representative images of HE staining, Sirius Red staining, and immunohistochemical staining for Col1, α-SMA, and MFAP4 in liver tissues from WT+Control, KO+Control, WT+TAA, and KO+TAA groups after 12 weeks of treatment. Quantification of the positive staining areas is shown below (n = 6 mice/treatment groups). qPCR analysis of the expression levels of Col1a1, Acta2, and Timp1 in total mRNA extracted from liver tissues of CCl 4 ( E ) and TAA ( F ) induced fibrotic mice (n = 6 mice/treatment groups). ( G ) Western blot analysis of the expression levels of total Col1, α-SMA, and TIMP1 in liver tissues from Mfap4 +/+ and Mfap4 -/- mice in the Oil- or CCl 4 -induced liver fibrosis model (n = 6 mice/treatment groups). ( H ) Western blot analysis of the expression levels of total Col1, α-SMA, and TIMP1 in liver tissues from Mfap4 +/+ and Mfap4 -/- mice in the control- or TAA-induced liver fibrosis model (n = 6 mice/treatment groups). Quantification of the positive staining areas: ∗Indicates a statistically significant difference between the WT+Oil and WT+CCl 4 groups or between the WT+Control and WT+TAA groups, with ∗∗∗ P < .001. #Indicates a statistically significant difference between the WT+CCl 4 and KO+CCl 4 groups or between the WT+TAA and KO+TAA groups, with ### P < .001. NS denotes no statistically significant difference between the WT+Oil and KO+Oil groups or between the WT+Control and KO+Control groups. ∗∗ P < .01, ∗∗∗ P < .001. Scale bars: 50 μm.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Staining, Immunohistochemical staining, Control, Expressing, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: The expression of MFAP4 is associated with fibrotic and apoptotic phenotypes. ( A ) Western blot analysis of Col1, α-SMA, and TIMP1 expression levels in whole cell lysates from LX-2 cells incubated with either control solvent DMSO or 2000 ng/mL rMFAP4 for 24 hours. Quantification of immunoblot results is shown on the right . ( B ) Western blot analysis of Col1, α-SMA, TIMP1, and MFAP4 expression levels in whole cell lysates from LX-2 cells transfected with either control vector or OE-MFAP4 plasmid for 72 hours. Quantification of immunoblot results is shown on the right . ( C ) Western blot analysis of Col1, α-SMA, TIMP1, and MFAP4 expression levels in whole cell lysates from LX-2 cells treated with control Scramble, Scramble+TGFβ1, Si-MFAP4, and Si-MFAP4+TGFβ1 for 72 hours. Quantification of immunoblot results is shown on the right . ( D, E ) CCK-8 assay was performed to measure the cell proliferation after treatment with OE-MFAP4 for various time points or different concentrations of rMFAP4. ( F, G ) Cell migration ability after OE-MFAP4 or after cells were treated with rMFAP4. ( H, I ) Western blot analysis of Bcl-2, Cleaved caspase-3, and Bax protein levels in total LX-2 cell lysates after OE-MFAP4 or Si-MFAP4. The Bcl-2/Bax ratio and the relative expression levels of Cleaved caspase-3 are presented. ( J, K ) Representative images of flow cytometry analysis showing the levels of apoptosis in LX-2 cells after OE-MFAP4 or Si-MFAP4. In the knockdown experiment: ∗indicates a statistically significant difference between the Scramble and Scramble+TGFβ1 groups, #indicates a statistically significant difference between the Si-MFAP4 and Si-MFAP4+TGFβ1 groups, and ˆindicates a statistically significant difference between the Scramble and Si-MFAP4 groups. Scale bars: 50 μm in ×40 magnification, 25 μm in ×100 magnification. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Expressing, Western Blot, Incubation, Control, Solvent, Transfection, Plasmid Preparation, CCK-8 Assay, Migration, Flow Cytometry, Knockdown

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: si-MFAP4 knockdown efficiency, and MFAP4 promotes HSCs proliferation and intrahepatic localization. ( A ) Validation of siRNA knockdown efficiency. qPCR and WB validation of Control group, Negative control group, and 3 MFAP4-knockdown siMFAP4 groups. NS denotes no statistically significant difference between the Control and Scramble groups. ∗∗ P < .01, ∗∗∗ P < .001. ( B ) CCK-8 assay showing proliferative changes of LX-2 cells treated with BSA negative control versus rMFAP4. ( C ) CCK-8 assay showing proliferative changes of LX-2 cells treated with TGF-β1 positive control versus rMFAP4. NS denotes no statistically significant difference between the BSA and treatment groups. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001. ( D ) Representative immunofluorescence images showing colocalization of ALB (hepatocyte marker, red ) and MFAP4 ( green ) in CCl 4 -induced fibrotic mouse liver. Scale bars = 200 μm. ( E ) Immunofluorescence analysis of α-SMA (activated HSCs marker, red ) and MFAP4 ( green ) in CCl 4 -induced fibrotic liver tissues. Scale bars = 200 μm.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Knockdown, Biomarker Discovery, Control, Negative Control, CCK-8 Assay, Positive Control, Immunofluorescence, Marker

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: HSCs-derived MFAP4 plays a central regulatory role in liver fibrosis. ( A ) In LX-2 cells, TGF-β1 stimulation significantly up-regulated MFAP4, COL1A1, and ACTA2 mRNA levels in a time-dependent manner. NS denotes no statistically significant difference between the Control and TGF-β1 groups. NS denotes no statistically significant difference between the Control and β groups. ∗∗ P < .01, ∗∗∗ P < .001. ∗∗∗ P < .001 versus untreated controls. ( B ) In TGF-β1-treated THLE-2 cells, MFAP4 expression showed minimal changes. ∗ P < .05, ∗∗∗ P < .001 versus untreated controls. ( C ) Western blot analysis confirmed baseline MFAP4 expression in HSCs and hepatocytes, with TGF-β1-induced MFAP4 up-regulation specifically observed in HSCs. ( D ) Schematic diagram of the Transwell coculture system for hepatocyte-HSCs interaction studies. ( E ) Western blot analysis of cocultured HSCs demonstrated that MFAP4 knockdown in HSCs (vs hepatocytes) significantly reduced fibrotic markers (α-SMA, Col1)and MFAP4 expression.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Derivative Assay, Control, Expressing, Western Blot, Knockdown

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: Increased expression of integrin αvβ3 in fibrotic liver tissue and activated HSCs, with MFAP4 binding to integrin αvβ3 on activated HSCs. ( A, B ) Western blot analysis showing up-regulation of integrin αvβ3 expression in fibrotic liver tissue, ( C ) and increased expression of integrin αvβ3 in activated HSCs. Coimmunoprecipitation (Co-IP) assays. IP was performed using antibodies against integrin αv ( D ), integrin β3 ( E ), and MFAP4 ( F ) to capture the respective protein complexes. ( I ) Double immunofluorescence staining for MFAP4 and integrin αv or β3 in activated LX-2 cells. Scale bars: 25 μm. ( G ) Double immunofluorescence staining for MFAP4, α-SMA, and integrin αv or β3 in CCl 4 -induced fibrotic liver tissue. ( H ) Double immunofluorescence staining for MFAP4, α-SMA, and integrin αv or β3 in TAA-induced fibrotic liver tissue. Scale bars: 50 μm.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Expressing, Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Double Immunofluorescence Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: The partial reversal of the MFAP4-induced phenotype by pathway inhibitors. The fibrotic and apoptotic phenotypes induced by rMFAP4 incubation in LX-2 cells and by MFAP4 overexpression in LX-2 cells can be partially reversed by the integrin αvβ3 inhibitors Cilengitide (1 μM) ( A-D ) and Cyclo(-RGDfK) (2 μM) ( G-H ). Quantitative analysis of Western blotting is shown on the right . ∗Indicates a statistical significance between the rMFAP4 or OE-MFAP4 group and the DMSO or OE-Vector group; #Indicates statistical significance between the rMFAP4 or OE-MFAP4 group and the rMFAP4 + inhibitor group or OE-MFAP4 + inhibitor group. ∗∗ P < .01, ∗∗∗ P < .001. # P < .05, ## P < .01, ### P < .001.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Incubation, Over Expression, Western Blot, Plasmid Preparation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: The relationship between MFAP4 and the activation of the FAK/PI3K/NFκB signaling pathway. ( A, B ) The integrin αvβ3 inhibitor Cilengitide (1 μM) partially reverses the activation of the FAK/PI3K/NFκB signaling pathway induced by rMFAP4 incubation or OE-MFAP4 in LX-2 cells. ( C, D ) Another integrin αvβ3 inhibitor, Cyclo(-RGDfK) (2 μM), partially reverses the activation of the FAK/PI3K/NFκB signaling pathway induced by rMFAP4 incubation or OE-MFAP4 in LX-2 cells. ( E, F ) The FAK-specific inhibitor Defactinib hydrochloride (10 μM) partially reverses the activation of the FAK/PI3K/NFκB signaling pathway induced by rMFAP4 incubation or OE-MFAP4 in LX-2 cells. Quantification of immunoblot results is shown on the right . ( G ) Immunofluorescence showing nuclear translocation of p-P65 in LX-2 cells after 24 hours of rMFAP4 incubation. Scale bars: 25 μm.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: Activation Assay, Incubation, Western Blot, Immunofluorescence, Translocation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: In vivo showing that the absence of MFAP4 alleviates the activation of the FAK/PI3K/NFκB signaling. ( A, B ) Activation of the FAK/PI3K/NFκB signaling pathway in CCl 4 and TAA-induced fibrotic livers. Quantification of immunoblot results is shown on the right (n = 6 mice/treatment groups). ( C, D ) Reduced activation of the FAK/PI3K/NFκB pathway in fibrotic livers of Mfap4 -/- mice compared to Mfap4 +/+ mice. Quantification of immunoblot results is shown on the right (n = 6 mice/treatment groups). ( E ) Immunofluorescence analysis of p-P65 nuclear translocation in CCl 4 or TAA-induced fibrotic livers. ( F ) Double immunofluorescence staining of αSMA and Cleaved Caspase3 for analysis of HSC apoptosis in fibrotic liver of Mfap4 +/+ and Mfap4 −/− mice. Scale bars: 25 μm ( E ) or 200 μm ( F ). ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: In Vivo, Activation Assay, Western Blot, Immunofluorescence, Translocation Assay, Double Immunofluorescence Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: MFAP4 Deficiency Attenuates Liver Fibrosis by Regulating Hepatic Stellate Cell Fate Through Inhibition of the FAK/PI3K/NFκB Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101548

Figure Lengend Snippet: Apoptosis detection in HSCs from fibrotic liver. TUNEL assay for apoptosis detection and α-SMA immunofluorescence analysis of HSCs apoptosis in fibrotic livers of and Mfap4 +/+ and Mfap4 -/- mice. Scale bars: 50 μm.

Article Snippet: Recombinant human MFAP4 protein: (HY- P77993 , MCE).

Techniques: TUNEL Assay, Immunofluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Maturation of the Mfa1 Fimbriae in the Oral Pathogen Porphyromonas gingivalis

doi: 10.3389/fcimb.2018.00137

Figure Lengend Snippet: ELISA of binding between post-Rgp processed Mfa3 and other Mfa proteins. Rgp-processed forms of Mfa1 or Mfa4, along with recombinant Mfa2 or Mfa5 (1 μg) were immobilized on a microtiter plate. BSA was used as a control. Binding of Rgp-processed Mfa3 at the concentrations indicated was detected with antibodies to Mfa3 (1:5,000) followed by secondary anti-rabbit IgG HRP-linked antibodies (1:5,000). Signals were developed with the TMB substrate and absorbance values were measured at 450 nm. The binding data were analyzed using a 1:1 saturation binding model, and K D and R 2 values are shown. The data are from 3 independent experiments.

Article Snippet: Recombinant Mfa proteins or BSA (1 μg each) were immobilized on a 96-well microplate (Corning).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant

Journal: Clinical and Translational Medicine

Article Title: Integrative multiomics analysis of human atherosclerosis reveals a serum response factor‐driven network associated with intraplaque hemorrhage

doi: 10.1002/ctm2.458

Figure Lengend Snippet: Biological extension of the results of the multiomics analysis. (A) Correlation‐based heatmap shows correlations and corresponding p ‐values (denoted as asterisk) between multiomics components 1–4 (derived from the transcriptomic block) and plaque traits. Specifically, correlations of components with collagen content and αSMA – PDGFRα + fibroblast‐like cell content are shown as scatter plots (non‐IPH, n = 16; IPH, n = 26; all plaques, n = 42). (B) Transcription factors (TFs) targeting the elements from the first component of multiomics sPLS‐DA‐based set of genes/proteins/peptides were extracted by iRegulon with default settings. Likely interactions of TFs with their downstream targets as obtained from iRegulon are indicated by directed lines with different color per TF. Genes, proteins and peptides, and TFs are depicted in different shapes; gene‐level differential expression (log2 fold change) is shown by green‐red gradient. The top five enriched TFs for multiomics component 1 ranked by normalized enrichment score (NES) are shown below, with the same colors as the directed lines in the network. (C) SRF network operating in plaques primarily steers the MKL1/2, not the ELF4 coregulated pathway. The p ‐values were calculated by hypergeometric test. (D) Representative pictures of IHC for MFAP4 (brown area) in non‐IPH and IPH plaques. Scale bar = 100 μm; 100× magnification. (E) Differences of IHC‐based MFAP4 positive area (%) between paired non‐IPH and IPH plaques (both n = 13)

Article Snippet: After cooling down for 20 min, sections were incubated overnight at 4°C with polyclonal rabbit anti‐human MFAP4 antibody (1:1000, NBP2‐30439: Novus Biologicals).

Techniques: Derivative Assay, Blocking Assay, Quantitative Proteomics

Journal: iScience

Article Title: Highly efficient generation of self-renewing trophoblast from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110874

Figure Lengend Snippet:

Article Snippet: Anti-MFAP5 Antibody , Atlas Antibodies , HPA010553.

Techniques: Purification, Produced, Virus, Control, Recombinant, Lysis, Extraction, Cell Culture, Gene Expression, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Western Blot, Library Quantification, Multiplex Assay, Sequencing, Software, Modification

Journal: The EMBO Journal

Article Title: Developmental beta-cell death orchestrates the islet’s inflammatory milieu by regulating immune system crosstalk

doi: 10.1038/s44318-024-00332-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: To generate the Tg(mfap4: turquoise2) line, the pDEST mfap4:turquoise2 vector purchased from Addgene (#135218) was injected in single-cell zebrafish embryos.

Techniques: Recombinant, Sequencing, Plasmid Preparation, TUNEL Assay, Glucose Assay, Software

Journal: Nature communications

Article Title: Calcium Dependent FAK/CREB/TNNC1 Signaling Mediates the Effect of Stromal MFAP5 on Ovarian Cancer Metastatic Potential

doi: 10.1038/ncomms6092

Figure Lengend Snippet: Identification of MFAP5 as one of the most highly overexpressed proteins in ovarian CAFs with clinical Significance. ( A ) Heat map showing 819 significantly differentially expressed genes in microdissected CAFs and ovarian epithelial tumor samples obtained from 33 patients, normal stromal fibroblasts obtained from 8 normal ovaries, and 6 normal ovarian surface epithelial tissue samples. ( B ) Quantitative RT-PCR analysis of RNA isolated from microdissected normal ovarian cortical fibroblasts, and the stromal fibroblastic (S: green bars) and epithelial (T: red bars) components from the same patients are shown. Normal fibroblasts did not have MFAP5 expression. Cancer stromal fibroblasts had significantly higher levels of MFAP5 expression than did those in the epithelial components. ( C ) Immunolocalization of MFAP5 in advanced HGSC samples with high (a) and low (b) levels of MFAP5 expression in the cancer stroma. S, cancer stroma; T, epithelial component. Bar = 100 µm. ( D ) Western blot analysis demonstrating higher levels of MFAP5 expression in CAFs than in NOFs and ovarian cancer cells. Relative normalized protein expression levels are shown. ( E ) Immunolocalization of MFAP5 was performed on 130 advanced HGSC tissues samples to determine the clinical relevance of stromal MFAP5 expression and HGSC patient survival. Kaplan-Meier analysis revealed that low stromal MFAP5 expression was significantly associated with a longer overall survival duration than patients who have high stromal MFAP5 expression (p < 0.001; log-rank test). Patients with high (above the median) and low (at or below the median) stromal MFAP5 expression levels had a median overall survival duration of 24 months and 54 months respectively.

Article Snippet: Pre-designed human MFAP5 (Hs00185803_m1), TNNC1 (Hs00896999_g1), cyclophilin A (Hs99999904_m1), and murine Mfap5 (Mm00489404_m1) TaqMan gene expression assays (Life Technologies Corp., Grand Island, NY) were used in the PCR.

Techniques: Quantitative RT-PCR, Isolation, Expressing, Western Blot

Journal: Nature communications

Article Title: Calcium Dependent FAK/CREB/TNNC1 Signaling Mediates the Effect of Stromal MFAP5 on Ovarian Cancer Metastatic Potential

doi: 10.1038/ncomms6092

Figure Lengend Snippet: Secretory proteins upregulated in CAFs compared to HOSE, NOFs and ovarian cancer epithelia

Article Snippet: Pre-designed human MFAP5 (Hs00185803_m1), TNNC1 (Hs00896999_g1), cyclophilin A (Hs99999904_m1), and murine Mfap5 (Mm00489404_m1) TaqMan gene expression assays (Life Technologies Corp., Grand Island, NY) were used in the PCR.

Techniques:

Journal: Nature communications

Article Title: Calcium Dependent FAK/CREB/TNNC1 Signaling Mediates the Effect of Stromal MFAP5 on Ovarian Cancer Metastatic Potential

doi: 10.1038/ncomms6092

Figure Lengend Snippet: Stromal MFAP5-induced ovarian cancer cell motility and invasion potential in vitro and in vivo . ( A ) Relative motility of ovarian cancer cells when co-cultured with CAF793092 primary ovarian CAFs in the presence of different antibodies was studied. Control IgG, and anti-α5 antibodies did not affect cancer cell motility, while anti-αVβ3, and anti-MFAP5 antibodies attenuated cancer cell motility in the co-culture system (mean ± SD of 3 independent experiments; p < 0.001; two-tailed Student t -test). ( B ) A series of images obtained from treatment of OVCA432 cells with recombinant MFAP5 protein in an temperature controlled imaging chamber for 48 hours was analyzed using the MetaMorph software program for tracking of the movement of individual cells. Presence of exogenous MFAP5 enhanced OVCA432 cell motility. ( C ) Relative motility of cancer cells was evaluated after treatment with recombinant MFAP5 in the presence of control IgG, anti-α5 or anti-αVβ3 antibodies compared with antibody-free controls, the presence of anti-αVβ3 antibodies abrogated MFAP5-induced cell motility (mean ± SD of 3 independent experiments; p < 0.01; two-tailed Student t -test). ( D ) Invasion assay using Matrigel invasion chamber showed that recombinant MFAP5 enhanced ovarian cancer cell invasiveness (mean ± SD of 3 independent experiments; p < 0.001; two-tailed Student t -test). ( E ) A schematic diagram showing the procedures of using intraperitoneally implanted Matrigel plugs to evaluate the effects of MFAP5 on invasion potential of ovarian cancer cells. ( F and G ) Immunolocalization of MFAP5 (red signal) and cytokeratin 18 (brown signal) on paraffin sections Matrigel plugs collected from the peritoneal cavities of mice; Bar = 100 µm. The invaded cancer cells with cytokeratin 18 staining were quantified using the Image-Pro Plus software program (mean ± SD; n = 10 Matrigel plugs per treatment group for each cell line; p = 0.0002 and 0.0162 for OVCA432 and A224 cells respectively; Mann-Whitney U test). Staining for MFAP5 showed that MFAP5 positive cells were found only in Matrigel plugs embedded with MFAP5 overexpressing fibroblasts, suggesting that stromal MFAP5 promoted ovarian cancer cell invasion into the plugs implanted. ( H and I ) Immunolocalization of p-ERK1/2 and troponin C in paraffin sections of Matrigel plugs collected from the peritoneal cavities of mice demonstrating that the effects of stromal MFAP5 on cancer cell motility and invasion potential were mediated by upregulation of p-ERK1/2 and troponin C expression; Bar = 100 µm and 50 µm respectively.

Article Snippet: Pre-designed human MFAP5 (Hs00185803_m1), TNNC1 (Hs00896999_g1), cyclophilin A (Hs99999904_m1), and murine Mfap5 (Mm00489404_m1) TaqMan gene expression assays (Life Technologies Corp., Grand Island, NY) were used in the PCR.

Techniques: In Vitro, In Vivo, Cell Culture, Control, Co-Culture Assay, Two Tailed Test, Recombinant, Imaging, Software, Invasion Assay, Staining, MANN-WHITNEY, Expressing

Journal: Nature communications

Article Title: Calcium Dependent FAK/CREB/TNNC1 Signaling Mediates the Effect of Stromal MFAP5 on Ovarian Cancer Metastatic Potential

doi: 10.1038/ncomms6092

Figure Lengend Snippet: In vivo stromal Mfap5 silencing reduced ovarian tumor growth and metastasis. ( A ) A schematic diagram showing the procedures of using intraovarian tumor injection model to evaluate the effects of MFAP5 on invasion and metastatic potential of ovarian cancer cells. ( B ) Luciferase labeled ovarian cancer cells were injected into left ovary of nude mice and luminescence was measured at week 6. Luciferase activities were significantly weaker in stromal Mfap5 silenced groups when compared to the control siRNA groups (N = 9–10 per treatment group). ( C ) Reduced metastatic spread of cancer cells within the abdominal cavity was observed in the Mfap5 siRNA encapsulated chitosan nanoparticles treated groups when compared to the control group. ( D ) Significantly reduced number of metastatic tumor nodules, tumor weight and ascites volume were observed in the Mfap5 knockdown groups when compared with the control siRNA group after necropsy (mean ± SD; n = 9–10 per treatment group; p < 0.001; Mann-Whitney U test). (E) Primary tumor burden was quantified by measuring the primary tumor area in the ovaries using images of H&E tissue sections prepared from tumors harvested from the control scramble siRNA treatment groups and two Mfap5-targeting siRNA treatment groups. Similar primary tumor burdens were observed in the control and Mfap5-targeted animal groups based on Mann-Whitney U tests. ( F ) Tumor tissues were fixed in formalin and embedded with paraffin followed by immunolocalization of Mfap5 to confirm the successful knockdown of stromal Mfap5 expression. S, Stroma; T, Tumor; Bar = 100 µm.

Article Snippet: Pre-designed human MFAP5 (Hs00185803_m1), TNNC1 (Hs00896999_g1), cyclophilin A (Hs99999904_m1), and murine Mfap5 (Mm00489404_m1) TaqMan gene expression assays (Life Technologies Corp., Grand Island, NY) were used in the PCR.

Techniques: In Vivo, Injection, Luciferase, Labeling, Control, Knockdown, MANN-WHITNEY, Expressing

Journal: Nature communications

Article Title: Calcium Dependent FAK/CREB/TNNC1 Signaling Mediates the Effect of Stromal MFAP5 on Ovarian Cancer Metastatic Potential

doi: 10.1038/ncomms6092

Figure Lengend Snippet: Contribution of calcium-dependent signaling pathway activation, TNNC1 overexpression, and F-actin cytoskeleton reorganization to MFAP5-stimulated ovarian cancer cell motility. ( A ) Heat map of motility-promoting genes associated with calcium signaling whose expression was upregulated in MFAP5-treated OVCA432 cells. ( B ) Interacting network of motility-promoting and calcium signaling-related genes whose expression was upregulated at least 1.5-fold in MFAP5-treated OVCA432 cells created using the Ingenuity Pathway Analysis software program (Ingenuity Systems, Redwood City, California). ( C ) Upregulation of TNNC1 mRNA expression in five serous ovarian cancer cell lines after MFAP5 treatment (mean ± SD of 3 independent experiments; p < 0.001; two-tailed Student t -test). ( D ) Abrogation of MFAP5’s stimulatory effect on ovarian cancer cell motility by knockdown of TNNC1 expression using TNNC1 -targeting siRNAs and a scrambled non-targeting siRNA as control (mean ± SD of 3 independent experiments; two-tailed Student t -test). ( E ) MFAP5-induced F-actin rearrangement and activated stress fiber formation in A224 and ALST cells; Bar = 10 µm.

Article Snippet: Pre-designed human MFAP5 (Hs00185803_m1), TNNC1 (Hs00896999_g1), cyclophilin A (Hs99999904_m1), and murine Mfap5 (Mm00489404_m1) TaqMan gene expression assays (Life Technologies Corp., Grand Island, NY) were used in the PCR.

Techniques: Activation Assay, Over Expression, Expressing, Software, Two Tailed Test, Knockdown, Control

Journal: Nature communications

Article Title: Calcium Dependent FAK/CREB/TNNC1 Signaling Mediates the Effect of Stromal MFAP5 on Ovarian Cancer Metastatic Potential

doi: 10.1038/ncomms6092

Figure Lengend Snippet: Effects of MFAP5 on motility are mediated via calcium mobilization and increasing traction force in ovarian cancer cells. ( A ) Attenuation of the stimulatory effect of MFAP5 on ovarian cancer cell motility by the cell-permeant calcium chelator BAPTA/AM but not the solvent control DMSO (mean ± SD of 3 independent experiments; p = 0.013 and < 0.001 for A224 and ALST cells respectively; two-tailed Student t -test). ( B ) Abrogation of activated stress fiber formation and actin reorganization in A224 and ALST cells pretreated with BAPTA/AM but not those pretreated with DMSO; Bar = 10 µm. ( C ) The mean normalized time course of Ca 2+ mobilization in A224 and ALST cells induced by MFAP5. Hank’s balanced salt solution (HBSS) containing MFAP5 or the HBSS alone was added to the bath of cells at t = 0 (mean ± SEM of 4 independent experiments for each treatment group). ( D ) Cell traction force maps of A224 and ALST cells before and after exposure to MFAP5. ( E ) The normalized total traction force exerted by A224 and ALST cells induced by MFAP5. Dotted grey lines are SEM. Only one side of SEM is shown. Purple lines are regression lines. HBSS containing MFAP5 or HBSS alone was added to the bath of cells at t = 120 s. MFAP5 significantly increases the slope of regression line compared to that of HBSS in both cell lines (p < 0.05; two-tailed Student t -test). N = number of cells used for the normalized time course.

Article Snippet: Pre-designed human MFAP5 (Hs00185803_m1), TNNC1 (Hs00896999_g1), cyclophilin A (Hs99999904_m1), and murine Mfap5 (Mm00489404_m1) TaqMan gene expression assays (Life Technologies Corp., Grand Island, NY) were used in the PCR.

Techniques: Solvent, Control, Two Tailed Test

Journal: Nature communications

Article Title: Calcium Dependent FAK/CREB/TNNC1 Signaling Mediates the Effect of Stromal MFAP5 on Ovarian Cancer Metastatic Potential

doi: 10.1038/ncomms6092

Figure Lengend Snippet: Western blot analysis of key signaling molecules implicated in calcium-dependent pathways and TNNC1 transcriptional regulation. Western blot analyses on proteins isolated from MFAP5-treated A224 and ALST ovarian cancer cells showed that, ( A ) Treatment of exogenous MFAP5 increased p-FAK (Y861), p-PLC-γ1 (Y783), p-PKCθ (T538), p-ERK1/2 (T202/Y204), p-CREB (S133), total Jun, and p-Jun (S63, S73 and S243) expressions in A224 ovarian cancer cells. ( B ) Treatment of exogenous MFAP5 increased p-FAK (Y861), p-PLC-γ1 (Y783), p-PKCθ (T538), p-ERK1/2 (T202/Y204), p-CREB (S133), total Jun, and p-Jun (S63, S73 and S243) expressions in ALST ovarian cancer cells. Relative normalized protein expression levels with respect to the corresponding control were presented. ( C ) A graphical summary of the molecular signaling events involved in MFAP5-mediated ovarian cancer cell motility and invasiveness. ( D ) The presences of FAK, ERK or CBP/CREB interaction inhibitors abrogated the motility promoting effects of MFAP5 in both A224 and ALST ovarian cancer cells (mean ± SD of 3 independent experiments; p < 0.001; two-tailed Student t -test).

Article Snippet: Pre-designed human MFAP5 (Hs00185803_m1), TNNC1 (Hs00896999_g1), cyclophilin A (Hs99999904_m1), and murine Mfap5 (Mm00489404_m1) TaqMan gene expression assays (Life Technologies Corp., Grand Island, NY) were used in the PCR.

Techniques: Western Blot, Isolation, Expressing, Control, Two Tailed Test

Journal: Nature communications

Article Title: Calcium Dependent FAK/CREB/TNNC1 Signaling Mediates the Effect of Stromal MFAP5 on Ovarian Cancer Metastatic Potential

doi: 10.1038/ncomms6092

Figure Lengend Snippet: Correlation between tumor TNNC1 and stromal MFAP5 expression in human HGSC samples revealed the clinical relevance of TNNC1 to ovarian cancer patient survival. ( A ) Abrogation of MFAP5-induced increase in TNNC1 mRNA expression in A224, ALST, and OVCA432 cells after pretreatment with the cell-permeant calcium chelator BAPTA/AM, a CBP/CREB inhibitor, or a c-Jun inhibitor (mean ± SD of 3 independent experiments; two-tailed Student t -test). ( B ) Immunolocalization of TNNC1 and MFAP5 in advanced-stage HGSC tumor sections demonstrating a positive correlation between high stromal MFAP5 and high tumor TNNC1 expression. S, stroma; T, tumor. Bar = 100 µm. ( C ) Positive correlation between stromal MFAP5 expression and tumor TNNC1 expression in HGSC tumors (N = 107; r = 0.515; p < 0.001; Spearman’s Correlation). ( D ) Kaplan-Meier analysis of the clinical significance of tumor TNNC1 expression with 107 FFPE tumor samples obtained from HGSC patients. Low tumor TNNC1 expression was significantly correlated with improved overall patient survival (N = 107; p = 0.018; log-rank test).

Article Snippet: Pre-designed human MFAP5 (Hs00185803_m1), TNNC1 (Hs00896999_g1), cyclophilin A (Hs99999904_m1), and murine Mfap5 (Mm00489404_m1) TaqMan gene expression assays (Life Technologies Corp., Grand Island, NY) were used in the PCR.

Techniques: Expressing, Two Tailed Test