Journal: Theranostics

Article Title: Reversal of bleomycin-induced rat pulmonary fibrosis by a xenograft of human umbilical mesenchymal stem cells from Wharton's jelly

doi: 10.7150/thno.33741

Figure Lengend Snippet: Transplantation of HUMSCs reduced the activation of fibroblasts and promoted the expression of TLR-4 in the left lungs of rats with PF. (A) Left lung sections were labeled with anti-α-SMA antibody to indicate activated fibroblasts in the left lungs of each group. (B) Western blot images represent the contents of α-SMA in the rats' left lungs. Quantitative results of α-SMA from western blotting are shown. The results indicated that activated fibroblasts significantly increased from Day 7 to 49. The activated fibroblasts significantly decreased after the transplantation of high doses of HUMSCs. (C) Rat Acta 2 mRNA were extracted from left lung suspensions and analyzed by quantitative RT-PCR, demonstrating that a significant decrease in acta2 expression in the BLM+HUMSCs (HD) group on Day 49. (D) Cell number in bronchoalveolar lavage fluid (BALF) of the BLM+HUMSCs (HD) group reduced significantly compared with that in the BLM group on Day 49. (E) The immunohistostaining of anti-TLR-4 showed that some TLR-4 positive cells were localized in connective tissue (arrow heads), however, some in the alveoli (arrows). (F) To further explore whether TLR-4 expression was localized in M2 macrophage, the left lung tissue sections from the BLM+HUMSCs (HD) group on Day 49 were subjected to double-staining with anti-TLR-4 (green) and anti-CD163 (red) antibodies. (G) The contents of TLR-4 in the rats' left lungs were detected through western blotting. The quantitative results indicated that TLR-4 significantly increased following the transplantation of HUMSCs. (H) Quantitative analysis of real-time RT-PCR of rat TLR-4 mRNA in the left lung tissue lysate demonstrated that rat TLR-4 mRNA was increased in the BLM+HUMSCs (HD) group (n = 3/group). (I) The result of RT-PCR of human TLR-4 revealed that human TLR-4 was only found in cell lysate of A549 cell line, but not in the lungs of all groups. (J) Left lung samples from the Normal, BLM, and BLM+HUMSCs (HD) groups on Day 49 were subjected to Human Cytokine Antibody Array analysis. The results suggested that the transplantation of HUMSCs increased human FGF-6 and IGF-1 concentrations in rats with pulmonary fibrosis. (K) Analyses were conducted using Rat Cytokine Antibody Array. The results indicated that the transplantation of HUMSCs stimulated rats with pulmonary fibrosis to produce higher amounts of β-NGF, fractalkine, and GM-CSF in their left lungs. ✱ vs the Normal group, p < 0.05. # vs the BLM group, p < 0.05. ♠ vs the BLM+HUMSCs (LD) group, p < 0.05. ⧫ vs the BLM+HUMSCs (HD) group, p < 0.05.

Article Snippet: To elucidate which human cytokines released from HUMSCs were involved in the treatment of rat PF, a human protein cytokine kit (AAH-CYT-2000, RayBio Human Cytokine Antibody Array C Series 2000, RayBiotech) was used to screen the expression of 174 human cytokines (n = 3/group).

Techniques: Transplantation Assay, Activation Assay, Expressing, Labeling, Western Blot, Quantitative RT-PCR, Double Staining, Reverse Transcription Polymerase Chain Reaction, Ab Array

Journal: Molecules

Article Title: Polyphenol Effects on Splenic Cytokine Response in Post-Weaning Contactin 1-Overexpressing Transgenic Mice

doi: 10.3390/molecules24122205

Figure Lengend Snippet: Levels of IL-12 in splenic supernatants from cell cultures of postnatal day 8 pups. Abbreviations: wt = wild type; +/+ = homo, +/− = hetero; PMA: phorbol myristate acetate, POP: suckling pups that received milk from dams administered with polyphenols. Evaluation of cytokines was performed using a cytometric bead array (CBA) mouse soluble protein Flex Set kit, as described in the Materials and Methods section. Statistical analysis was performed using Bonferroni’s test for comparison between groups. GraphPad Prism statistical software release 5.0 for Windows Vista was used. Statistical significance was set at p < 0.05.

Article Snippet: Cell supernatants obtained from single mice were incubated with the cytometric bead array (CBA) mouse soluble protein Flex Set kit (Becton Dickinson, Milan, Italy).

Techniques: Comparison, Software

Journal: The Journal of Biological Chemistry

Article Title: Urokinase-type Plasminogen Activator (uPA) Promotes Angiogenesis by Attenuating Proline-rich Homeodomain Protein (PRH) Transcription Factor Activity and De-repressing Vascular Endothelial Growth Factor (VEGF) Receptor Expression *

doi: 10.1074/jbc.M115.678490

Figure Lengend Snippet: Functional consequences of uPA binding to HHEX. A, TranSignal Transcription factor Protein Array. Array Membranes (Panomics, Version I) spotted in duplicate with proteins expressed from full-length transcription factor cDNAs were incubated with scuPA (10 nm) for 2 h. Bound scuPA was detected with rabbit anti-uPA polyclonal antibodies, HRP-conjugated secondary goat anti-rabbit antibodies, and chemiluminescence substrate. B, co-immunoprecipitation (co-IP) of uPA and HHEX from 293HEK cells ectopically expressing HHEX-FLAG and uPA. HEK 293 cells were transfected with HHEX-FLAG in pcDNA3.1 and uPA/pcDNA3.1 vectors. Two days later, cells were harvested, and nuclear extracts were prepared using the NucBuster protein extraction kit (Novagen). uPA or HHEX was immunoprecipitated using mouse monoclonal anti-uPA or anti-FLAG antibodies immobilized on agarose beads. Normal mouse Ig immobilized on agarose beads was used as the negative control. Immune complexes were analyzed by Western blot (WB) using anti-uPA rabbit polyclonal antibodies and HRP-conjugated anti-FLAG mouse monoclonal antibodies. C, co-immunoprecipitation of uPA and HHEX from the nuclear extracts of human scuPA-treated human LMVECs. uPA and HHEX were co-immunoprecipitated from the nuclear extracts using mouse monoclonal anti-uPA and anti-HHEX antibodies, respectively. Normal mouse Ig immobilized on agarose beads was used as the negative control. Immune complexes were analyzed by Western blot using anti-uPA rabbit polyclonal antibodies and anti-HHEX rabbit polyclonal antibody. D, binding of HHEX to recombinant uPA variants. Recombinant uPA variants (33 nm in PBS) or BSA (1%) as the negative control were immobilized in 96-well plates in triplicate and incubated with nuclear extract from HEK293 cells transfected with pcDNA 3.1/HHEX-FLAG. Bound HHEX-FLAG was detected using an anti-HHEX polyclonal antibody/HRP-anti-rabbit antibody sandwich ELISA. Optical density was read at 450 nm (OD450). y axes denote normalized OD450 obtained by subtracting the OD450 measured in BSA-coated wells from the OD450 measured in wells coated with uPA variants. E, uPA inhibits binding of HHEX to the target DNA sequence. HEK 293 cells were transfected with HHEX-FLAG in pcDNA3.1. Two days later, cells were harvested, and nuclear extracts were prepared. EMSA reactions were performed using biotinylated double-stranded HHEX specific oligonucleotides and corresponding unlabeled oligonucleotide. 1, no nuclear extracts (NE); 2, + NE in presence of 50× excess unlabeled HHEX-specific oligonucleotide; 3, + NE alone; 4, + NE in the presence of BSA; 5, + scuPA (500 ng); 6, + NE + scuPA (500 ng); 7, + scuPA in the presence of anti-uPA antibody; 8, + NE + mouse IgG; 9, + NE + anti-FLAG mouse monoclonal antibody; 10, + NE in the presence of irrelevant unlabeled oligonucleotides. S, probe shift caused by HHEX overexpression. SS. probe supershift caused by HHEX-bound anti-FLAG antibody. F, VEGFR1 and VEGFR2 promoter luciferase reporter assay. Human VEGFR1 or VEGFR2 promoter-driven luciferase reporter pGL3 (51) vectors were co-transfected in EA.hy926 cells with uPA- and HHEX-encoding pcDNA3.1 vectors alone or in combination. Luciferase activity was measured at 24 h using a Dual Luciferase Reporter assay kit (Promega). Outcomes were normalized to the activity of co-transfected renilla luciferase-encoding pRL-CMV vector (Promega).

Article Snippet: Transcription Factor Protein Binding Array Analysis Binding of uPA to transcription factors was analyzed using the TranSignal TM TF Protein Array kit (Version I, catalog #MA3501, Panomics) per the manufacturer's instructions as described by us previously ( 38 ).

Techniques: Functional Assay, Binding Assay, Protein Array, Incubation, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Transfection, Protein Extraction, Negative Control, Western Blot, Recombinant, Sandwich ELISA, Sequencing, Over Expression, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation