Journal: Biochimica et biophysica acta

Article Title: Dependence of fibroblast infiltration in tumor stroma on type IV collagen-initiated integrin signal through induction of platelet-derived growth factor.

doi: 10.1016/j.bbamcr.2015.02.004

Figure Lengend Snippet: Fig. 5. Correlation of type IV-α1 collagen and PDGF-A expression in human cancer cells. (A) Five-μm paraffin-embedded tissue sections of glioma and hepatoma were prepared, deparaffinized, and dual-immunofluorescence stained for type IV collagen and PDGF-A with appropriate antibodies. Arrows indicate that type IV collagen and PDGF-A were colocalized within the tumor section. (B) The U118MG- and Huh7-derived shCol cells were established as described in Fig. 1. The transcripts of PDGF-A in U118MG- and Huh7-derived cells were deter- mined by quantitative real time PCR. (C) Type IV collagen and PDGF-A proteins were detected by Western blot analysis. GAPDH served as the loading control. (D) Fibroblast recruitment assay was done using human fibroblast MRC-5 cells in the conditions described in Fig. 3. Conditioned media collected from U118MG- and Huh7-derived cells supplemented with or without 10 ng/ml PDGF-A growth factor were used as attractants. (E) The conditioned media collected from U118MG and Huh7 parental cells were pretreated with 20 μg/ml neutralizing anti- PDGF-A Ab or control Ab and then used as attractants and loaded in the lower chamber. Shown are the averages of three independent experiments. *: p b 0.05 compared with control cells.

Article Snippet: Rabbit antibodies (Abs) for type IV collagen and PDGF-A were obtained from Novus Biologicals LLC (Littleton, CO).

Techniques: Expressing, Staining, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Life

Article Title: Lysophosphatidic Acid Modulates TGF-β2-Induced Biological Phenotype in Human Conjunctival Fibroblasts

doi: 10.3390/life14060770

Figure Lengend Snippet: LPA-induced effects on gene expression of ECM proteins COL1 , COL4 , COL6 , FN , and αSMA in 2D and 3D cultured HconF cells. The 2D and 3D HconF cells that had been incubated with 5 ng/mL TGF-β2 and/or 500 nM LPA in addition to non-treated control cells were subjected to qPCR analysis. Measurements were conducted in triplicate using fresh preparations (2D: n = 5; 3D: n = 10 spheroids each). * p < 0.05, ** p < 0.01, and *** p < 0.005 (ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: Briefly, 4% paraformaldehyde-fixed 3D spheroids were incubated in 3% BSA in PBS for 3 h. After washing twice with PBS for 30 min each time, 3D spheroids were sequentially exposed to 1:200 dilution of primary antibodies, including an anti-human COL1 (#600-401-103-0.1, ROCKLAND Antibodies & Assays, Limerick, PA, USA), COL4 (#600-401-106S, ROCKLAND Antibodies & Assays, Limerick, PA, USA), COL6 (#009-001-108, ROCKLAND Antibodies & Assays, Limerick, PA, USA), and FN (#A-11, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) rabbit antibody at 4 °C overnight.

Techniques: Gene Expression, Cell Culture, Incubation, Control, Comparison

Journal: Life

Article Title: Lysophosphatidic Acid Modulates TGF-β2-Induced Biological Phenotype in Human Conjunctival Fibroblasts

doi: 10.3390/life14060770

Figure Lengend Snippet: Immunolabeling of ECM proteins COL1 , COL4 , COL6 , FN , and αSMA of the 3D HconF spheroids. The 3D HconF spheroids that had been incubated with 5 ng/mL TGF-β2 and/or 500 nM LPA in addition to non-treated control cells were subjected to immunocytochemistry analysis. Analyses were conducted in triplicate using fresh preparations ( n = 5). Relative staining intensities of the molecules are plotted in the right panels. * p < 0.05 (ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: Briefly, 4% paraformaldehyde-fixed 3D spheroids were incubated in 3% BSA in PBS for 3 h. After washing twice with PBS for 30 min each time, 3D spheroids were sequentially exposed to 1:200 dilution of primary antibodies, including an anti-human COL1 (#600-401-103-0.1, ROCKLAND Antibodies & Assays, Limerick, PA, USA), COL4 (#600-401-106S, ROCKLAND Antibodies & Assays, Limerick, PA, USA), COL6 (#009-001-108, ROCKLAND Antibodies & Assays, Limerick, PA, USA), and FN (#A-11, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) rabbit antibody at 4 °C overnight.

Techniques: Immunolabeling, Incubation, Control, Immunocytochemistry, Staining, Comparison