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

Journal: Hypertension

Article Title: Calpain-1 Regulation of Matrix Metalloproteinase 2 Activity in Vascular Smooth Muscle Cells Facilitates Age-Associated Aortic Wall Calcification and Fibrosis

doi: 10.1161/hypertensionaha.112.196840

Figure Lengend Snippet: Figure 3. Overexpression of calpain-1 induces collagen type I, II, and III and reduces elastin expression levels in vascular smooth muscle cells (VSMC). A, Representative Western blots of collagen I and collagen III in VSMC after infection by GFP or CANP1 adenoviruses for 48 hours. B, Photomicrographs (×400) of collagen II protein staining (brown color) from young (8-month) and old (30-month) rat aortic walls. Nuclei are counterstained with hematoxylin (blue color). L indicates lumen; M, media. C and D, Representative Western blots and average protein levels of collagen II (C) and elastin (D) in young VSMC infected with GFP or CANP1 for 48 hours. *P<0.05 compared with GFP-infected 8-month VSMC, n=3.

Article Snippet: Primary antibodies used for immunostaining (IS) and Western blotting (WB) Antibody Species Titer for IS Titer for WB Source -actin Mouse 1:10000 Sigma-Aldrich Co. LLC, St. Louis, MO calpain-1 Rabbit 1:100 1:2500 Sigma-Aldrich Co. LLC, St. Louis, MO collagen I Rabbit 1:1000 Rockland Antibodies & Assays, Gilbertsville, PA collagen II Rabbit 1:50 1:1000 Abcam, Cambridge, MA collagen III Rabbit 1:1000 Rockland Antibodies & Assays, Gilbertsville, PA elastin Rabbit 1:1000 Millipore, Billerica, MA MMP2 Mouse 1:50 1:1000 Millipore, Billerica, MA MT1MMP Rabbit 1:1000 Millipore, Billerica, MA osteonectin Rabbit 1:1000 Millipore, Billerica, MA osteoponin Rabbit 1:1000 Millipore, Billerica, MA Smad 2/3 Mouse 1:1000 Cell Signaling Technology, Inc, Danvers, MA TGF-1 Rabbit 1:500 Santa Cruz Biotechnology, Inc, Santa Cruz, CA TIMP2 Goat 1:500 R&D Systems, Inc. Minneapolis, MN 9 Figure S1: Interactions between calpain-1 and MMP2.

Techniques: Over Expression, Expressing, Western Blot, Infection, Staining

Journal: Hypertension

Article Title: Calpain-1 Regulation of Matrix Metalloproteinase 2 Activity in Vascular Smooth Muscle Cells Facilitates Age-Associated Aortic Wall Calcification and Fibrosis

doi: 10.1161/hypertensionaha.112.196840

Figure Lengend Snippet: Figure 6. Overexpression of calpain-1 regulates extracellular matrix remodeling, particularly fibrosis and vascular calcification in cultured carotid artery rings. Representative Western blot of tissue inhibitor of metalloprotease 2 (TIMP2), calpain-1, elastin, transforming growth factor-β1 (TGFβ1), (A) Smad 2/3, collagen I, II, III, (B) and osteopontin (OPN) and osteonectin (ON) (C) in cultured carotid artery rings isolated from 8-month-old rat after infection by GFP or CANP1 or TIMP2 adenoviruses for 48 hours.

Article Snippet: Primary antibodies used for immunostaining (IS) and Western blotting (WB) Antibody Species Titer for IS Titer for WB Source -actin Mouse 1:10000 Sigma-Aldrich Co. LLC, St. Louis, MO calpain-1 Rabbit 1:100 1:2500 Sigma-Aldrich Co. LLC, St. Louis, MO collagen I Rabbit 1:1000 Rockland Antibodies & Assays, Gilbertsville, PA collagen II Rabbit 1:50 1:1000 Abcam, Cambridge, MA collagen III Rabbit 1:1000 Rockland Antibodies & Assays, Gilbertsville, PA elastin Rabbit 1:1000 Millipore, Billerica, MA MMP2 Mouse 1:50 1:1000 Millipore, Billerica, MA MT1MMP Rabbit 1:1000 Millipore, Billerica, MA osteonectin Rabbit 1:1000 Millipore, Billerica, MA osteoponin Rabbit 1:1000 Millipore, Billerica, MA Smad 2/3 Mouse 1:1000 Cell Signaling Technology, Inc, Danvers, MA TGF-1 Rabbit 1:500 Santa Cruz Biotechnology, Inc, Santa Cruz, CA TIMP2 Goat 1:500 R&D Systems, Inc. Minneapolis, MN 9 Figure S1: Interactions between calpain-1 and MMP2.

Techniques: Over Expression, Cell Culture, Western Blot, Isolation, Infection

Journal: International journal of cosmetic science

Article Title: Moisturizing and antioxidant factors of skin barrier restoring cream with shea butter, silkflo and vitamin E in human keratinocyte cells.

doi: 10.1111/ics.13014

Figure Lengend Snippet: FIGURE 5 Effect of MZ on procollagen type 1 level. Values are expressed as average of triplicate experiment and are represented as mean ± SEM. * represent significant difference from H2O2 induced group (p < 0.05), # represent significant difference from untreated cells.

Article Snippet: Estimation of type I procollagen The level of type I procollagen was measured using a human type I procollagen ELISA kit (Elabscience Biotechnology, USA).

Techniques: