hla-g1 cdna  (GenScript corporation)

Journal: PLoS ONE

Article Title: Long-Lasting Inhibitory Effects of Fetal Liver Mesenchymal Stem Cells on T-Lymphocyte Proliferation

doi: 10.1371/journal.pone.0019988

Figure Lengend Snippet: HLA-G was detected in BM-MSC and FL-MSC by using RT-PCR, western blot and flow cytometry. ( a ) The RT-PCR products from BM-MSC and FL-MSC, as a result of using specific primers for HLA-G1 and HLA-G5, were analyzed on an agarose gel. A FON-57 cell line and β-actin were used as positive controls for HLA-G1 and HLA-G5, and as a loading control, respectively. In other experiments, M8-G1 and M8-G5 transfected melanoma cell lines were used as positive control. ( b ) Detection of HLA-G isoforms in BM-MSC (passage 2–4, n = 3), FL-MSC (passage 12–15, n = 3), M8-G5 or M8-G1 (positive control) by western blot with 4H84 mAb. The 39 kDa bands correspond to HLA-G1 and the 37 kDa to the soluble form (the soluble HLA-G5 or the shed HLA-G1). ( c ) The same MSC were also stained at the cell surface or intracellularly with MEM-G/9-PE (filled histograms), a specific anti-HLA-G antibody. ( d ) Dot blot analysis showed that MSC do not secretes soluble HLA-G (clone 5A6G7), compared to melanoma M8-G5 cell line used as positive control. ( e ) To induce the shedding of the membrane bound HLA-G1, cells were treated or not with PMA (10 ng/ml) for 12 h and their HLA-G expression was evaluated by flow cytometry. PMA induce the release of HLA-G from M8-G1 cells as well as BM-MSC or FL-MSC, also confirmed by ELISA assay ( f ). Histogram indicated the percentage of decrease compared to the non treated cells (* P <0.01; ** P <0.005). Averages of three independent experiments are shown.

Article Snippet: M8 cells were transfected with a full-length HLA-G5 cDNA (M8-HLA-G5) or HLA-G1 cDNA (M8-HLA-G1) subcloned in vector pcDNA (Invitrogen Life Technologies) and used as positive control for HLA-G5 and HLA-G1 expression, respectively.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Agarose Gel Electrophoresis, Transfection, Positive Control, Staining, Dot Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Long-Lasting Inhibitory Effects of Fetal Liver Mesenchymal Stem Cells on T-Lymphocyte Proliferation

doi: 10.1371/journal.pone.0019988

Figure Lengend Snippet: ( a ) Western blot analysis (4H84 and 5A6G7 mAbs) for HLA-G1 (39 kDa) and HLA-G5 (37 kDa) isoform after MLR (4 days). Following MLR, lymphocytes and MSC (passage 2–4 and 12–15 for adult and fetal cells, respectively) were loaded separately. ( b ) Soluble HLA-G released by MSC after MLR (4 days) was evaluated by ELISA assay. Two PBL and four adult or fetal MSC were used to evaluate HLA-G secretion. M8-G1 and M8-G5 melanoma cells were used as positive control for HLA-G1 and HLA-G5 isoform protein (contained and secreted, respectively. ( c ) CFSE-labeled T cells were cultured with MSC (ratio T∶MSC 5∶1) directly (cell-cell contact) or in transwell culture system. Neutralizing anti-HLA-G antibody (87G mAb) was added on days 0 and 2 (20 µg/ml), as previously reported . ( d ) BM-MSC lacks immunomodulatory properties after more than 8 passages of culture, whereas FL-MSC have a more prolonged inhibitory effect on T cells proliferation which remains HLA-G dependant. ( e ) Western blot analysis of HLA-G1 (4H84 mAb) protein expression in BM-MSC (passage 3 and 10) compared to FL-MSC (passage 10 and 25). Averages of three independent experiments are shown. Error bars represent the SD. P- values were calculated using Student's t-test. * P <0.05, ** P <0.01; *** P <0.001, ns = not statistically significant.

Article Snippet: M8 cells were transfected with a full-length HLA-G5 cDNA (M8-HLA-G5) or HLA-G1 cDNA (M8-HLA-G1) subcloned in vector pcDNA (Invitrogen Life Technologies) and used as positive control for HLA-G5 and HLA-G1 expression, respectively.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control, Labeling, Cell Culture, Expressing