Journal: Biomarker Research

Article Title: Human induced-T-to-natural killer cells have potent anti-tumour activities

doi: 10.1186/s40364-022-00358-4

Figure Lengend Snippet: Immunophenotypic characteristics of ITNK cells. A Representative CyTOF analysis of the immunophenotypic profiles of CB-derived T cells on Day 2 to Day 10 post electroporation with sg BCL11B . B, C UMAP plots with colored circles highlighting the cells at different time points (Day 2, Day 4, Day 6, Day 8, and Day 10) ( B ) and different clusters of T cells based on their immunophenotypic profiles ( C ). D Representative T cell marker (CD4, CD8A, CD62L, CCR7, and γδTCR) and NK cell-associated marker (CD56, NKp30, NKp44, NKp46, CD11c and CD16) expression in various subtypes of T cells and ITNKs. E Representative flow cytometric detection of CCR1, CCR3, CCR6, CCR8, and CXCR4 in T cells (CD3 + CD4 + /CD8 + ), ITNKs (CD3 + CD4 + NKp30+/CD3 + CD8 + NKp46 + ) and normal NK cells (CD3 − CD56 + ). Data are representative of three independent experiments. F Graph summarizing the percentages of CCR1, CCR3, CCR6, CCR8, and CXCR4 cells in T cells, ITNKs and NK cells after 14 days of culture. The results represent the mean ± SD. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001; one-way ANOVA with Tukey’s multiple comparisons test

Article Snippet: T cells were enriched from mononuclear leukocytes with CliniMACS CD4 reagent (200–070-132, Miltenyi Biotec, Germany) and CliniMACS CD8 reagent (200–070-115, Miltenyi Biotec, Germany) and activated by MACS GMP T cell transaction (170–076-156, Miltenyi Biotec, Germany) for 72 h according the manufacture’s protocols.

Techniques: Derivative Assay, Electroporation, Marker, Expressing

Journal: Biomarker Research

Article Title: Human induced-T-to-natural killer cells have potent anti-tumour activities

doi: 10.1186/s40364-022-00358-4

Figure Lengend Snippet: ITNKs acquire transcriptional profiles of NK cells. A UMAP visualization of sg BCL11B -transduced T cells after batch correction, colored by samples collected for scRNA-seq analysis on Day 2 (D2), Day 4 (D4), Day 6 (D6), Day 8 (D8), and Day 10 (D10) post electroporation. B UMAP visualization of sg BCL11B -transduced T cells mainly classified into 6 different clusters based on their gene expression profiles. C Plots showing the expression levels of selected genes associated with T (C D3E, CD8A and CD4 ) or NK cell lineages ( NCAM1, NCR3, ID2, IL2RB, and NFIL3 ) in sg BCL11B -transduced T cells based on scRNA-seq analysis. D Violin plots showing the expression levels of NK cell- and T cell-associated genes, AP-1 family genes, glycolysis-associated genes and genes regulating proliferation in activated T cells (Cluster 0 (CD4 T) and Cluster 3 (CD8 T)), effector T cells (Cluster 1 (CD4 T) and Cluster 4 (CD8 T)), and ITNKs (Cluster 2 (CD4 ITNK) and Cluster 5 (CD8 ITNK)). E T cells and ITNKs were purified (purity > 90%) from sgCtrl- and sg BCL11B -electroporated T cells from Donors 1 and 2. Purified NK cells (CD3 − CD56 + ) (purity > 90%) were enriched from NK cell cultures from Donors 1–3. Cells from Donors 1–3 were collected from CB samples. Principal component analysis (PCA) was used to evaluate the similarities in global gene expression profiles among purified ITNKs, NK cells, and T cells. F Differential expression patterns of selected NK cell-, glycolysis-, and T cell-associated genes and AP-1 family genes differentially expressed in ITNKs, NK cells, and T cells based on RNA-seq analysis

Article Snippet: T cells were enriched from mononuclear leukocytes with CliniMACS CD4 reagent (200–070-132, Miltenyi Biotec, Germany) and CliniMACS CD8 reagent (200–070-115, Miltenyi Biotec, Germany) and activated by MACS GMP T cell transaction (170–076-156, Miltenyi Biotec, Germany) for 72 h according the manufacture’s protocols.

Techniques: Electroporation, Gene Expression, Expressing, Purification, Quantitative Proteomics, RNA Sequencing

Journal: Biochemical Journal

Article Title: Involvement of miR-30c and miR-301a in immediate induction of plasminogen activator inhibitor-1 by placental growth factor in human pulmonary endothelial cells

doi: 10.1042/bj20101585

Figure Lengend Snippet: Figure 1 PlGF down-regulates the levels of miRNA-30c and miR-99a, leading to increased PAI-1 mRNA expression

Article Snippet: Reagents were obtained as follows: human recombinant PlGF was from R&D Systems; HIF-1α siRNA (small interfering RNA), PlGF siRNA and corresponding control scRNA (scrambled RNA), primary antibodies against PAI-1 and β-actin, and secondary antibodies conjugated to HRP (horseradish peroxidase) were from Santa Cruz Biotechnology; the c-Jun TranSilent siRNA vector was from Panomics; and actinomycin D was from Enzo Life Sciences.

Techniques: Expressing

Journal: Biochemical Journal

Article Title: Involvement of miR-30c and miR-301a in immediate induction of plasminogen activator inhibitor-1 by placental growth factor in human pulmonary endothelial cells

doi: 10.1042/bj20101585

Figure Lengend Snippet: Figure 2 PlGF-induced PAI-1–3′-UTR reporter activity is abrogated by miR-30c and miR-99a, but not by miR-99a

Article Snippet: Reagents were obtained as follows: human recombinant PlGF was from R&D Systems; HIF-1α siRNA (small interfering RNA), PlGF siRNA and corresponding control scRNA (scrambled RNA), primary antibodies against PAI-1 and β-actin, and secondary antibodies conjugated to HRP (horseradish peroxidase) were from Santa Cruz Biotechnology; the c-Jun TranSilent siRNA vector was from Panomics; and actinomycin D was from Enzo Life Sciences.

Techniques: Activity Assay

Journal: Biochemical Journal

Article Title: Involvement of miR-30c and miR-301a in immediate induction of plasminogen activator inhibitor-1 by placental growth factor in human pulmonary endothelial cells

doi: 10.1042/bj20101585

Figure Lengend Snippet: Figure 3 miR-30c and miR-301a affect PlGF-induced PAI-1 mRNA and PAI-1 protein levels

Article Snippet: Reagents were obtained as follows: human recombinant PlGF was from R&D Systems; HIF-1α siRNA (small interfering RNA), PlGF siRNA and corresponding control scRNA (scrambled RNA), primary antibodies against PAI-1 and β-actin, and secondary antibodies conjugated to HRP (horseradish peroxidase) were from Santa Cruz Biotechnology; the c-Jun TranSilent siRNA vector was from Panomics; and actinomycin D was from Enzo Life Sciences.

Techniques:

Journal: The FASEB Journal

Article Title: Placental growth factor negatively regulates retinal endothelial cell barrier function through suppression of glucose-6-phosphate dehydrogenase and antioxidant defense systems

doi: 10.1096/fj.201901353R

Figure Lengend Snippet: Recombinant PlGF has specific affinity with its mAb (PL5D11D4) and disrupts HREC barrier function. A, B) Dot immunoassay demonstrated the specific affinity of PlGF antibody (PL5D11D4) with rbPlGF (A) and rhPlGF (B). rhVEGF and BSA acted as negative controls. HRP, horseradish peroxidase. C) HRECs were cultured and grown with endothelial growth medium 2 (EGM-2) (with growth factors such as VEGF-A). After the cells grew to confluence, the culture medium was changed to endothelial cell growth basal medium 2 (EBM-2) (without growth factors), and PBS control and rhPlGF protein (100 ng/ml) were added to the culture medium and incubated for at least 2 d. TEER was monitored by an ECIS system at an AC frequency of 4 KHz in real time. TEER curves of HRECs that were treated with rhPlGF protein and PBS control. Error bars represent sd out of 4 duplicate samples. The experiments were repeated at least 3 times. D) WB result for VE-cadherin, β-catenin, and ZO-1. β-Actin was used as the protein loading control. E, F) Immunofluorescence staining results of tight junction ZO-1 and adhesion protein VE-cadherin. ****P < 0.0001.

Article Snippet: Cell treatments and small interfering RNA transfection After HRECs or BRECs gained ∼80–90% confluence, the culture medium was replaced with fresh medium with the following desired treatment agents: d -glucose (25 mM), l -glucose (25 mM), mannitol (25 mM), anti-PlGF antibody (PL5D11D4), anti-VEGFR1 antibody (MF1; ImClone Systems, New York, NY, USA), anti-VEGFR2 antibody (DC101; ImClone Systems), rhPlGF protein (264-PGB-010/CF; R&D Systems, Minneapolis, MN, USA), rhVEGF/PlGF heterodimers (297-VP-005/CF; R&D Systems), VEGF-165 (293-VE-010/CF; R&D Systems), mouse IgG, peroxiredoxin (PRDX)6 inhibitor (MJ33 lithium salt, 1007476-63-2; Cayman Chemicals, Ann Arbor, MI, USA), and the glucose-6-phosphate dehydrogenase (G6PD) inhibitor dehydroepiandrosterone (DHEA; MilliporeSigma, Burlington, MA, USA).

Techniques: Recombinant, Cell Culture, Control, Incubation, Immunofluorescence, Staining

Journal: The FASEB Journal

Article Title: Placental growth factor negatively regulates retinal endothelial cell barrier function through suppression of glucose-6-phosphate dehydrogenase and antioxidant defense systems

doi: 10.1096/fj.201901353R

Figure Lengend Snippet: PlGF regulates G6PD, PRDX6, and EC barrier function through VEGFR1 and VEGFR2 signaling. A) WB results for G6PD. HRECs that were treated with rhPlGF and VEGF-165 protein (100 ng/ml each, for 2 d) were used for the WB assay: β-actin was used as the protein loading control. Note that rhPlGF, but not VEGF-165, down-regulated the protein level of G6PD compared with the PBS control. B) Double immunofluorescence labeling of G6PD and PRDX6. The HRECs that were treated with the PBS control, rhVEGF(a), and rhPlGF protein were fixed and stained with G6PD (green) and PRDX6 (red). The DAPI staining of the nucleus (blue) was used as the counterstaining. Note that the 2 proteins were colocalized in the PBS-treated and rhVEGF-treated cells (yellow); however, the staining signals were reduced in the rhPlGF-treated cells. The complete images of all 3 fluorescent staining channels are shown in Supplemental Fig. S5. C) WB results of ZO-1, VE-cadherin, and β-catenin. The confluent HRECs were treated with PBS, PBS + rhPlGF (100 ng/ml), rhPlGF + VEGFR1 antibodies (100 µg/ml), rhPlGF + VEGFR2 antibody (100 µg/ml), and VEGFR1 antibody alone or VEGFR2 antibody alone for 2 d. Note that rhPlGF protein down-regulated the protein levels of ZO-1, VE-cadherin, and G6PD, which were prevented by the VEGFR1 or VEGFR2 antibody. The antibody alone did not change the protein levels, which were equivalent to those of the PBS control. D) WB results of ZO-1, VE-cadherin, β-catenin, occludin-1, claudin-5, and G6PD. The confluent HRECs were treated with the PBS, PlGF/VEGF heterodimer (50, 100, or 200 ng/ml), PlGF (100 ng/ml), or VEGF-165 (100 ng/ml). Note that PlGF, the PlGF/VEGF heterodimer (200 ng/ml), and VEGF-165 down-regulated ZO-1, VE-cadherin, β-catenin, and claudin-5 (but not occludin-1); however, only PlGF and PlGF/VEGF down-regulated G6PD. E) TEER (or resistance) of HRECs. After the HRECs grew to confluence, the PBS control, PlGF/VEGF, or VEGF-165 was added to the medium, and the TEER was measured with ECIS in real time. The TEER values of the PlGF/VEGF heterodimer (blue) and VEGF-165 (purple) were significantly lower than those of the PBS control (red). Error bars represent sd out of 4 duplicate samples. F) WB results of PlGF/VEGF heterodimers in HREC lysate and culture medium. The confluent HRECs were treated with PBS, 25 mM d-glucose [high glucose (HG)], and 25 mM l-glucose [normal glucose (NG)]. Note that compared with the PBS controls, the HG up-regulated PlGF/VEGF in the cell lysates but decreased the levels in the culture medium. The β-actin was presented in the HREC lysate (but not in the culture medium) and used as the protein loading control. ****P < 0.0001.

Article Snippet: Cell treatments and small interfering RNA transfection After HRECs or BRECs gained ∼80–90% confluence, the culture medium was replaced with fresh medium with the following desired treatment agents: d -glucose (25 mM), l -glucose (25 mM), mannitol (25 mM), anti-PlGF antibody (PL5D11D4), anti-VEGFR1 antibody (MF1; ImClone Systems, New York, NY, USA), anti-VEGFR2 antibody (DC101; ImClone Systems), rhPlGF protein (264-PGB-010/CF; R&D Systems, Minneapolis, MN, USA), rhVEGF/PlGF heterodimers (297-VP-005/CF; R&D Systems), VEGF-165 (293-VE-010/CF; R&D Systems), mouse IgG, peroxiredoxin (PRDX)6 inhibitor (MJ33 lithium salt, 1007476-63-2; Cayman Chemicals, Ann Arbor, MI, USA), and the glucose-6-phosphate dehydrogenase (G6PD) inhibitor dehydroepiandrosterone (DHEA; MilliporeSigma, Burlington, MA, USA).

Techniques: Control, Immunofluorescence, Labeling, Staining