Journal: Microorganisms

Article Title: Construction of a High-Expression System in Bacillus through Transcriptomic Profiling and Promoter Engineering

doi: 10.3390/microorganisms8071030

Figure Lengend Snippet: Bacterial strains and plasmids used in this work.

Article Snippet: The vector pSU03-AP allows to secretory expression of the alkaline protease (AprE) from B. pumilus BA06 in Bacillus [ ]. pMUTIN4 was used to provide the lac I gene [ ]. pGEM-T-HSA (Catalog No. HG10968-G) were purchased from Sino Biologic, Inc. (Beijing, China), which hosted the full cDNA encoding the human serum albumin (HSA).

Techniques: Expressing

Journal: International journal of molecular sciences

Article Title: β-Caryophyllene Inhibits Endothelial Tube Formation by Modulating the Secretome of Hypoxic Lung Cancer Cells-Possible Role of VEGF Downregulation.

doi: 10.3390/ijms25020810

Figure Lengend Snippet: Figure 6. Effect of BCP on VEGF-induced VEGFR2 phosphorylation in HUVECs. HUVECs were preincubated with BCP at selected concentrations (A) or at 10 µM (B) for 6 h, followed by stimulation with recombinant VEGF (10 ng/mL) for 5 min. In the case of the inhibitor experiment shown in (B), AM630 was added to the cells 30 min before BCP. All percentages shown refer to vehicle-treated HUVECs set at 100%. Data are mean ± SEM of n = 3 (A) or n = 4 (B) per group. Western blot images are representative of each experiment. * p ≤0.05, ** p ≤0.01 versus vehicle-treated HUVECs; one-way ANOVA with Dunnett’s (A) or Bonferroni’s (B) post hoc test.

Article Snippet: Recombinant human VEGF-165 (rVEGF, #HZ-1038) and recombinant human serum albumin (rHSA, #HZ-3001) were purchased from Proteintech (Planegg-Martinsried, Germany).

Techniques: Phospho-proteomics, Recombinant, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Exosomal PD-L1 functions as an immunosuppressant to promote wound healing

doi: 10.1080/20013078.2019.1709262

Figure Lengend Snippet: The sequences of sgRNA.

Article Snippet: In order to acquire establishing the stable cell line expressing PD-L1, SK-MEL-5 cells (human melanoma cell line) and B16F10 cells (mouse melanoma cell line) were infected with lentiviruses carrying human/mouse PD-L1 gene (C-OFP Spark tag) (Sino Biological inc).

Techniques:

Journal: Journal of Extracellular Vesicles

Article Title: Exosomal PD-L1 functions as an immunosuppressant to promote wound healing

doi: 10.1080/20013078.2019.1709262

Figure Lengend Snippet: The sequences of the qPCR primers.

Article Snippet: In order to acquire establishing the stable cell line expressing PD-L1, SK-MEL-5 cells (human melanoma cell line) and B16F10 cells (mouse melanoma cell line) were infected with lentiviruses carrying human/mouse PD-L1 gene (C-OFP Spark tag) (Sino Biological inc).

Techniques: Sequencing

Journal: Journal of Extracellular Vesicles

Article Title: Exosomal PD-L1 functions as an immunosuppressant to promote wound healing

doi: 10.1080/20013078.2019.1709262

Figure Lengend Snippet: Characterization of exosomes purified from melanoma cells (a). qPCR of PD-L1 mRNA levels in SK-MEL-5 cells ( WT ), PD-L1 knockout SK-MEL-5 cells ( Pd-l1 −/- ), IFN-γ treated cells ( WT+IFN-γ, Pd-l1 −/- +IFN-γ ) and PD-L1 overexpressing cells ( WT+PD-L1 ). n = 3. (b). Western blot for PD-L1, CD81, CD63, ALIX and GAPDH in the whole cell lysate (W) and purified exosomes (E) from WT, Pd-l1 −/- , WT+IFN-γ, Pd-l1 −/- +IFN-γ and WT+PD-L1 . (c). The protein yield of exosomes from WT, Pd-l1 −/- , WT+IFN-γ, Pd-l1 −/- +IFN-γ and WT+PD-L1 . n = 3. (d). TEM images of purified exosomes from WT, Pd-l1 −/- , WT+IFN-γ, Pd-l1 −/- +IFN-γ and WT+PD-L1 . Scale bar: 50 nm. (e-f). The size distribution (e) and the Zeta potential (f) of exosomes from WT, Pd-l1 −/- , WT+IFN-γ, Pd-l1 −/- +IFN-γ and WT+PD-L1 . n = 3. ***P < 0.001.

Article Snippet: In order to acquire establishing the stable cell line expressing PD-L1, SK-MEL-5 cells (human melanoma cell line) and B16F10 cells (mouse melanoma cell line) were infected with lentiviruses carrying human/mouse PD-L1 gene (C-OFP Spark tag) (Sino Biological inc).

Techniques: Purification, Knock-Out, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Exosomal PD-L1 functions as an immunosuppressant to promote wound healing

doi: 10.1080/20013078.2019.1709262

Figure Lengend Snippet: Exosomal PD-L1 suppressed T cell activation and promoted skin cell migration in vitro (a). Representative confocal image showed the appearance of exosomes as small-red dots. Scale bar: 5 μm. (b-c). Representative confocal images of pre-stained exosomes (red) colocalized with cell membrane (green) of HEK 293T (b) and Jurkat T cells (c). Scale bar: 5 μm. (d-e). Flow cytometry analysis of CFSE-labelled T cell proliferation assay. PBMCs (8 × 10 5 ) were incubated with WT (100 µg/mL)), Pd-l1 −/ − (100 µg/mL), WT+IFN-γ (100 µg/mL), Pd-l1 −/- +IFN-γ (100 µg/mL), WT+PD-L1 (100 µg/mL) and FK506 (100 nM) for 3 days and 7 days. Cells were cultured with mock reagents for 0, 3 and 7 days as Ctrl (d0), Ctrl (d3) and Ctrl (d7) , respectively. (f-g). Wound scratch assay of HDF cells and HaCaT cells. Wounded cells were incubated with WT (100 µg/mL), Pd-l1 −/- (100 µg/mL), WT+IFN-γ (100 µg/mL), Pd-l1 −/- +IFN-γ (100 µg/mL), WT+PD-L1 (100 µg/mL) and bFGF (2.5 ng/mL) for 24 h. Serum-free medium was used as control ( Ctrl ). 24 h migration rate (g) and representative wound images (f) of each group at 24 h were shown. Scale bar: 100 μm. n = 3. *P < 0.05; **P < 0.01; ns, not significant.

Article Snippet: In order to acquire establishing the stable cell line expressing PD-L1, SK-MEL-5 cells (human melanoma cell line) and B16F10 cells (mouse melanoma cell line) were infected with lentiviruses carrying human/mouse PD-L1 gene (C-OFP Spark tag) (Sino Biological inc).

Techniques: Activation Assay, Migration, In Vitro, Staining, Flow Cytometry, Proliferation Assay, Incubation, Cell Culture, Wound Healing Assay

Journal: Journal of Extracellular Vesicles

Article Title: Exosomal PD-L1 functions as an immunosuppressant to promote wound healing

doi: 10.1080/20013078.2019.1709262

Figure Lengend Snippet: Exosomal PD-L1 accelerated mouse skin wound closure (a). Representative wound images from mice treated with 20% PF-127 alone ( Ctrl ), treated with 20% PF-127 containing bFGF cytokine ( bFGF ), or 20% PF-127 containing exosomes ( WT, WT+PD-L1, WT+IFN-γ ) over 10 days of healing period. The same ring was used to compare the size of all wounds. (b). Comparison of the percentages of the open wound size over 10 days healing period between Ctrl, WT, WT+IFN-γ, WT+PD-L1 and bFGF groups. n = 3. (c). Representative histological images (HE) and immunohistochemical images for vimentin, α-SMA and Ki67 antibodies in the wounds on day 7. Scale bar: 200 μm. (d). qPCR of IL-6, TNF-α, granzyme B mRNA levels in the wounded skin on day 7 from different groups. n = 3. (e-h). Representative flow cytometry plots demonstrated change of CD4+ and CD8 + T cells (gated on positive CD3+ cells) in spleen (e) and peripheral lymph nodes (g) on day 7 of wound healing. (f) and (h) were quantitation from (e) and (g). n = 2–3. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Article Snippet: In order to acquire establishing the stable cell line expressing PD-L1, SK-MEL-5 cells (human melanoma cell line) and B16F10 cells (mouse melanoma cell line) were infected with lentiviruses carrying human/mouse PD-L1 gene (C-OFP Spark tag) (Sino Biological inc).

Techniques: Immunohistochemical staining, Flow Cytometry, Quantitation Assay