Journal: BMC Cancer

Article Title: CD26 Expression on T-Anaplastic Large Cell Lymphoma (ALCL) Line Karpas 299 is associated with increased expression of Versican and MT1-MMP and enhanced adhesion

doi: 10.1186/1471-2407-13-517

Figure Lengend Snippet: Confirmation of Versican expression in Karpas 299 cells and in CD26-depleted and Versican-depleted Karpas cells. A . Western blots confirmed versican expression in Karpas cell lines and clones resulting from knockdown of versican in parental Karpas 299 cells using shRNA. Whole cell lysates (30 μg) of Karpas, Dep1, Dep2, and two clones derived from knock down of versican in parental Karpas cells, 1A12 and 6RD3 were run on 7.5% gels. The top of the gel and 250 kD marker are indicated. Blots were probed with anti-versican antibody at 1:100 dilution, followed by anti-mouse HRP at 1:10,000 dilution. B . RT-PCR using V0 and V1 specific primers show product was present when RNA from the parental Karpas 299 cells was used but barely detectable when RNA from Dep1 or Dep2 was used as the template. Results from Western blots and RT-PCR were obtained from two independent experiments.

Article Snippet: Transduction ready viral particles for gene silencing of versican (versican shRNA, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used to infect Karpas cells at a ratio of 0.5 virus particles per cell.

Techniques: Expressing, Western Blot, Clone Assay, Knockdown, shRNA, Derivative Assay, Marker, Reverse Transcription Polymerase Chain Reaction

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Versican: a novel modulator of hepatic fibrosis.

doi: 10.1038/labinvest.2015.152

Figure Lengend Snippet: Figure 1 Versican expression in human liver cirrhosis and mouse liver fibrosis. (a) Sections of liver parenchyma from non-cirrhotic (n = 5) and cirrhotic (n = 5) human livers were stained using immunohistochemistry for versican (12C5), alongside normal (n = 5) and fibrotic (n = 5) mouse liver sections (after 4 weeks CCl4) (HPA004726). Representative non-cirrhotic human liver shown from a patient with metastatic colorectal carcinoma and representative cirrhotic human liver shown from a patient with primary sclerosing cholangitis. (b) Quantitation of versican immunostaining was performed with image thresholding and significance determined by two-way analysis of variance (*) (Po0.05). Mean value per lane is shown (n = 5) and error bars represent the s.e.m. CCL4, carbon tetrachloride.

Article Snippet: Data normalization relative to the 18 s RNA subunit was calculated using the comparative Ct (ΔΔCt) method to calculate the fold change between the control and experimental groups as previously described.77 Primer pairs used for the different mouse and human genes are listed in Table 1. siRNA Constructs and Cell Transfections The following expression plasmids were used: human versican siRNA (Santa Cruz cat. # sc-41903) and control siRNA (Santa Cruz cat. # sc-37007).

Techniques: Expressing, Staining, Immunohistochemistry, Quantitation Assay, Immunostaining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Versican: a novel modulator of hepatic fibrosis.

doi: 10.1038/labinvest.2015.152

Figure Lengend Snippet: Figure 3 Versican expression increases during liver fibrosis and reduces during recovery. (a) Total RNA was extracted from the livers of control mice, CCl4 4-week treated mice and mice recovering from CCl4-induced liver injury and analyzed by qPCR for versican and collagen-1 mRNA expression using 18 s RNA as endogenous control. (b) Liver injury and fibrogenesis were assessed in control mice for TNF-α, α-SMA, and TGF-β. Relative mRNA expression and s.e.m. values are shown. Significant differences determined with one-way analysis of variance are indicated compared with control (*) and compared with 4 weeks of treatment (†) (Po0.05, n = 9–10). CCL4, carbon tetrachloride; α-SMA, α-smooth muscle actin; TNF-α, tumor necrosis factor-α; TGF-β, transforming growth factor-β.

Article Snippet: Data normalization relative to the 18 s RNA subunit was calculated using the comparative Ct (ΔΔCt) method to calculate the fold change between the control and experimental groups as previously described.77 Primer pairs used for the different mouse and human genes are listed in Table 1. siRNA Constructs and Cell Transfections The following expression plasmids were used: human versican siRNA (Santa Cruz cat. # sc-41903) and control siRNA (Santa Cruz cat. # sc-37007).

Techniques: Expressing, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Versican: a novel modulator of hepatic fibrosis.

doi: 10.1038/labinvest.2015.152

Figure Lengend Snippet: Figure 4 CCl4-induced liver fibrosis leads to increase in α-SMA and versican V0 fragment. Total protein was extracted from liver samples of control, CCl4 4-week treated and CCl4-treated recovering mice. (a) Total protein samples were analyzed for mouse α-SMA expression using histone H3 as a protein normalizing control. (b and c) Total protein samples were analyzed for the ADAMTS generated versican fragments by western blot analysis using anti- DPEAAE neo-epitope antibody and β-actin as a protein normalizing control. Significant differences determined with one-way analysis of variance are indicated compared with control (*) and compared with 4 weeks of treatment (†) (Po0.05). Western blots shown demonstrate three representative lanes per time point. Data are representative of 3–4 repeat experiments. Mean band densitometry values per lane are shown (n = 9–10) and error bars represent the s.e.m. ADAMTS, A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs; CCL4, carbon tetrachloride; α-SMA, α-smooth muscle actin.

Article Snippet: Data normalization relative to the 18 s RNA subunit was calculated using the comparative Ct (ΔΔCt) method to calculate the fold change between the control and experimental groups as previously described.77 Primer pairs used for the different mouse and human genes are listed in Table 1. siRNA Constructs and Cell Transfections The following expression plasmids were used: human versican siRNA (Santa Cruz cat. # sc-41903) and control siRNA (Santa Cruz cat. # sc-37007).

Techniques: Control, Expressing, Generated, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Versican: a novel modulator of hepatic fibrosis.

doi: 10.1038/labinvest.2015.152

Figure Lengend Snippet: Figure 6 Activation of primary mouse stellate cells leads to increased versican expression. Primary HSCs were isolated from 3- to 6-week-old C57BL/6 mice as indicated in the methods. Isolated primary HSCs were activated by plastic adherence over 1, 3, 5, 7, 14, and 21 days. Total RNA was then extracted from cells and analyzed for (a) versican, (b) collagen-1, and (c) α-SMA mRNA expression using 18 s RNA as an endogenous control. (d) ADAMTS-1 and -4 were analyzed alongside TIMP-1 and -3 expression levels, using 18 S RNA as an endogenous control. Significant differences determined with one-way analysis of variance are indicated compared with day 1 (*) (Po0.05). Data are representative of 3–4 repeat experiments. Mean value per lane is shown (n = 3) and error bars represent the s.e.m. ADAMTS, A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs; HSC, hepatic stellate cell; α-SMA, α-smooth muscle actin; TIMP-1, tissue inhibitor of metalloproteinase-1.

Article Snippet: Data normalization relative to the 18 s RNA subunit was calculated using the comparative Ct (ΔΔCt) method to calculate the fold change between the control and experimental groups as previously described.77 Primer pairs used for the different mouse and human genes are listed in Table 1. siRNA Constructs and Cell Transfections The following expression plasmids were used: human versican siRNA (Santa Cruz cat. # sc-41903) and control siRNA (Santa Cruz cat. # sc-37007).

Techniques: Activation Assay, Expressing, Isolation, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Versican: a novel modulator of hepatic fibrosis.

doi: 10.1038/labinvest.2015.152

Figure Lengend Snippet: Figure 7 Transient knockdown of versican in LX2 cells leads to a decrease in versican V1 fragment and decreased HSC activation. Total RNA and protein were extracted from LX2 cells 48 h after transient knockdown of versican or not as indicated. Total RNA was analyzed for (a) collagen-1, (b) α-SMA, (c) KI67, and (d) TGF-β1 mRNA expression, respectively, by qPCR using 18 s RNA as an endogenous control. (e) Total protein extracts were analyzed for versican V1 fragment using β-actin as a protein normalizing control. (f) Total protein was extracted from LX2 cells after versican KD, staurosporine treatment or not as indicated and subjected to western blot analysis for PARP as indicated. (g) Total RNA was extracted from primary mouse HSC cultures 48 h after knockdown of versican or not (control) as indicated. mRNA expression was analyzed by qPCR for versican, α-SMA, collagen-1, and TGF-β1 using 18 s RNA as an endogenous control. Significant differences determined with one-way analysis of variance are indicated compared with no treatment control (*) (Po0.05). Data are representative of 3–4 repeat experiments. The mean band densitometry value per lane is shown (n = 3) in (e) and error bars represent the s.e.m. HSC, hepatic stellate cell; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; PARP, poly(ADP-ribose)polymerase.

Article Snippet: Data normalization relative to the 18 s RNA subunit was calculated using the comparative Ct (ΔΔCt) method to calculate the fold change between the control and experimental groups as previously described.77 Primer pairs used for the different mouse and human genes are listed in Table 1. siRNA Constructs and Cell Transfections The following expression plasmids were used: human versican siRNA (Santa Cruz cat. # sc-41903) and control siRNA (Santa Cruz cat. # sc-37007).

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