Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Characterization of thrombin generation after hepatic IRI and cytotoxicity of thrombin for hepatic SECs. (A) In the clinical study of patents who underwent hepatectomy (n = 14), plasma TAT levels were plotted at 2 time points: before and 1 day after hepatectomy. TAT levels were significantly elevated after operation. (B) In in vivo hepatic IRI models of mice, plasma TAT levels measured by ELISA was significantly increased after IRI compared with the naïve group (n = 6 in each group). (C‐a) In hepatic SECs cultured in the nonischemic condition, LDH cytotoxicity levels in the supernatant of cell cultures were elevated in a dose‐dependent manner with the addition of thrombin. The addition of 100 U/mL thrombin significantly increased LDH cytotoxicity compared with both groups of naïve and 10 U/mL thrombin (n = 4 in each group). (C‐b) In H‐R models of SECs in vitro, the addition of 100 U/mL thrombin also significantly increased LDH cytotoxicity compared with vehicle (n = 5 in each group). H‐R was established using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Clinical Proteomics, In Vivo, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Direct effect of dabigatran treatment on pure cultured hepatic SECs in an H‐R model in vitro. (A) In H‐R models of pure cultured hepatic SECs, dabigatran treatment significantly decreased LDH cytotoxicity levels in the supernatant compared with vehicle (n = 5 in each group). (B‐a) Dabigatran treatment significantly increased TM expression in cell lysate measured by ELISA compared with vehicle (n = 5 in each group). (B‐b) By contrast, in TM expression in the supernatant of cell cultures measured by ELISA, there was no significant difference between the vehicle and dabigatran groups (n = 5 in each group). (C‐a) Based on Western blot analysis, dabigatran treatment did not significantly affect HMGB‐1 expression in cell lysate compared with the vehicle group (n = 5 in each group). Quantification of HMGB‐1 band intensities normalized to β‐actin. (C‐b) By contrast, HMGB‐1 levels in the supernatant of cell cultures measured by ELISA were markedly reduced by dabigatran treatment compared with the vehicle (n = 5 in each group). In H‐R models of this study, SECs were cultured in a serum‐starved medium containing 100 U/mL thrombin and exposed to H‐R using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Cell Culture, In Vitro, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Direct effect of dabigatran treatment on pure cultured hepatocytes in an H‐R model in vitro. In H‐R models of pure cultured hepatocytes, there was no significant difference between the H‐R + vehicle group and the H‐R + dabigatran group (A) in LDH cytotoxicity levels in the supernatant of cell cultures, in TM levels in cell lysate measured by ELISA (B‐a), in TM levels in the supernatant of cell cultures measured by ELISA (B‐b), in HMGB‐1 expression in cell lysate evaluated by Western blot analysis (C‐a), or in HMGB‐1 levels in the supernatant of cell cultures measured by ELISA (C‐b) (n = 5 in each group). In Western blot analysis, quantification of HMGB‐1 band intensities normalized to β‐actin. In H‐R models of this study, cells were cultured in a serum‐starved medium containing 100 U/mL thrombin and exposed to H‐R using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Effects of dabigatran for hepatocytes in a paracrine communication model between hepatic SECs and hepatocytes. (A) A scheme of our paracrine communication model between hepatic SECs and hepatocytes. SECs were pretreated with or without dabigatran followed by incubation in a serum‐starved medium in the H‐R condition; thereafter the supernatant of the SECs was harvested. Hepatocytes were pretreated with the supernatant of SECs followed by incubation in a serum‐starved medium in the H‐R condition. (B) LDH cytotoxicity levels in the supernatant of hepatocyte cultures were significantly lower in the group of H‐R + the supernatant of SECs pretreated with dabigatran than in the group of H‐R + the supernatant of SECs pretreated with vehicle (n = 5 in each group). TUNEL staining detected cellular death of hepatocytes after H‐R (C‐a) in the group of H‐R + the supernatant of SECs pretreated with vehicle and (C‐b) in the group of H‐R + the supernatant of SECs pretreated with dabigatran (original magnification ×200). (C‐c) The supernatant of SECs pretreated with dabigatran markedly reduced the number of TUNEL‐positive hepatocytes compared with the supernatant of SECs pretreated with the vehicle (n = 5 in each group).

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Incubation, TUNEL Assay, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: Tumor-intrinsic RRBP1 inhibition triggers antitumor immunity. ( A ) Representative images of IHC staining for RRBP1 and CD8 + T cells in BC samples. ( B ) The correlation between RRBP1 expression and CD8 + T-cell infiltration was analyzed based on 96 patients from in-house BC cohort. Scale bar: 50 µm. ( C ) Representative images of IHC staining for RRBP1 expression in PD, SD, PR, and CR samples. Scale bar: 50 µm. ( D ) Bar plot showed the response rates of anti-PD-L1 therapy. Blue bars represent CR/PR, Red bars represent PD/SD. ( E ) Volcano plot of RNA-seq data for shNC or shRRBP1 tumors (n=3). Differentially expressed genes were identified with the threshold of |log2 (fold change) | >1 and FDR<0.05. ( F ) GSEA for DEGs showed the activation of immune-associated pathways in shRRBP1 tumors in the RNA-seq data. ( G ) Representative images of IHC and mIHC staining for RRBP1 and CD8 + T cells in shNC, shRRBP1, control or radezolid tumor tissues. Expression levels of the indicated proteins were displayed. Scale bar: 20 µm. ( H, I ) Flow cytometry showed the percentages of CD8 + T cells in CD3 + cells in shNC, shRRBP1, control or radezolid tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using Spearman correlation analysis ( B ), unpaired two-tailed t-test ( I ). ****p<0.0001. BC, bladder cancer; CR, complete response; FDR, false discovery rate; progressive disease; PR, partial response; PD-L1, programmed death-ligand 1; RNA-seq, RNA sequencing; RRB1, ribosomal-binding protein 1; SD, stable disease; IHC, immunohistochemistry; GSEA, gene set enrichment analysis; DEGs, differentially expressed genes; mIHC, multiplex immunohistochemistry.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Immunohistochemistry, Expressing, RNA Sequencing, Activation Assay, Staining, Control, Flow Cytometry, Two Tailed Test, Binding Assay, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: Single-cell RNA sequencing reveals the difference of CD8 + T-cell subgroup. The UMAP plot of CD8 + T cells subpopulation, color-coded by cell cluster and cell type. ( A ) The expression of markers in each CD8 + T cells subpopulation. ( B ) Bar plot showed the proportion of CD8 + T cells subpopulation in the shNC and shRRBP1 groups. ( C ) The percentage of each CD8 + T-cell clusters in shNC and shRRBP1 groups. ( D ) Heatmap showed the differentially activated pathway among all the CD8 + T-cell clusters. ( E ) The differentially expressed genes in CD8 + T cells between shNC and shRRBP1 groups. ( F ) KEGG analysis for differentially expressed genes showed the enrichment of immune-associated pathways. ( G, H ) mIHC and flow cytometric analysis displayed the tumor-infiltrating IFN-γ + or GZMB + CD8 + T cells in shNC or shRRBP1 tumor tissues. Scale bar: 20 µm. ( I–K ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Isotype control (IgG) or anti-mouse CD8 antibody administered on days –6, –3, and –1 before tumor challenge, with the same dose repeated on days 7, 9 and 11 after tumor challenge. Tumor sizes ( I ), volumes ( J ), and weight ( K ) were measured. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( H, K ) and two-way ANOVA with Tukey’s multiple comparison test ( J ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; GZMB, Granzyme B; IFN, interferon; TEX, exhausted T cells; UMAP, Uniform Manifold Approximation and Projection; mIHC, multiplex immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Single Cell, RNA Sequencing, Expressing, Injection, Control, Two Tailed Test, Comparison, Multiplex Assay, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: RRBP1 inhibition promotes antitumor immunity via the CXCL10-CXCR3 axis in BC. ( A ) ScRNA-seq data showed the CXCR3 expression of CD8+T cells in shNC and shRRBP1 groups. ( B ) The correlation between CXCR3 expression and CXCL10 expression or activated CD8 + T cell based on 571 patients from TCGA-BLCA cohort and GSE13507 cohorts. ( C ) MB49 cells were co-cultured with CD8 + T cells, and tumor cells were stained with crystal violet. ( D ) Evaluation of the effect of genetic inhibition of RRBP1 on the cytotoxicity of CD8 + T cells in vitro conditioned culture model. ( E ) Schematic diagram of in vitro CD8 + T-cell migration assays. ( F ) The number of CD8 + T cells passing through the membrane of a Transwell system was analyzed by flow cytometry. ( G–I ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice received intraperitoneal injection of either vehicle or anti-CXCL10 when the tumor volume reached a calculated average of 100 mm 3 . The tumor sizes ( G ), volumes ( H ), and weights ( I ) were measured. ( J ) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. ( K ) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, CXCR3 + CD8 + T cells, IFN-γ + CD8 + T cells or GZMB + CD8 + T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( D, F, I, K ) and two-way ANOVA with Tukey’s multiple comparison test ( H ). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; RRBP1, ribosomal-binding protein 1; scRNA-seq, single-cell RNA sequencing; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry; BLCA, bladder urothelial carcinoma.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Cell Culture, Staining, In Vitro, Migration, Membrane, Flow Cytometry, Injection, Two Tailed Test, Comparison, Binding Assay, Single Cell, RNA Sequencing, Immunohistochemistry, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: RRBP1 inhibition enhances response to anti-PD-L1 therapy in BC. ( A–D ) The protein expression of surface PD-L1 was analyzed in BC cells or tumor tissues by flow cytometry after RRBP1 inhibition and was shown as the mean fluorescence intensity. ( E–G ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice were received intraperitoneal injection of either vehicle or anti-PD-L1 antibody when the tumor volume reached a calculated average of 100 mm 3 . The tumor sizes ( E ), volumes ( F ), and weights ( G ) were measured. ( H ) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. ( I ) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, CXCR3 + CD8 + T cells, IFN-γ + CD8 + T cells or GZMB + CD8 + T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( B, D, G, I ) and two-way ANOVA with Tukey’s multiple comparison test ( F ). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; PD-L1, programmed death-ligand 1; RRBP1, ribosomal-binding protein 1; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Injection, Staining, Two Tailed Test, Comparison, Binding Assay, Immunohistochemistry, Multiplex Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 1. Tumor-associated soluble uPAR (s-uPAR) enhances HUVEC invasion, migration and angiogenesis. (a) Conditioned medium (CM) was collected from tumor cells (parental and stably expressing empty vector (EV), uPAR-cDNA (UR) and uPAR siRNA (UR-Si)). Immunoblot analyses were performed for s-uPAR and DDK using specific antibodies. (b) s-uPAR levels in CM were quantified using uPAR Quantikine Immunoassay kit. Columns: mean; bars: s.d.; n ¼ 3; *po0.01 vs parental control. (c) Cells were labeled (tumor cells: Qtracker-525-Green and HUVECs: Qtracker- 655-Red) and seeded into separate chambers of culture inserts. After 16 h, the culture inserts were removed and cells were allowed to migrate for a further 24 h. Images were captured at 0 and 24 h of incubation and cell migration was quantified using ImageJ software (NIH). The levels of HUVEC migration were normalized to HUVEC migration in parental cells and are represented as arbitrary units. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental control. (d) HUVEC invasion experiments were performed using ThinCertTM inserts as described in Materials and methods. The levels of HUVEC invasion was quantified and normalized to HUVEC invasion in parental-CM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM. (e, f) In vitro angiogenesis assay was performed as described in Materials and methods. The degree of angiogenic induction by CM was quantified by ImageJ software (NIH) for the numerical value of the product of the relative capillary length per microscopic field. Serum-free medium (SFM) and recombinant human uPAR (rh-uPAR) in SFM were used as controls (insets). Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **po0.01 vs UR-CM. uPAR antibody, uPAR-Ab; isotype control, NSp.IgG. (g) Migration assay was performed using CM. In this case, both chambers of culture inserts were seeded with HUVECs. After 16 h, the culture inserts were removed, CM was added and cells were allowed to migrate for 24 h. Invasion assay was performed as described above. uPAR-Ab. or Nsp.IgG were added to UR-CM before adding onto cells. rh-uPAR was added to SFM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Migration, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Labeling, Incubation, Software, In Vitro, Angiogenesis Assay, Recombinant, Invasion Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 2. s-uPAR recruits onto HUVEC membrane. Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. (a) HUVECs were cultured on CM for 24 h, labeled with anti-uPAR antibody, followed by Alexa Fluor-488-conjugated secondary antibody and were analyzed by fluorescence-activated cell sorting (FACS) for uPAR expression. Serum-free medium (SFM) and rh-uPAR were used as controls. Isotype control (Neg.). (b) HUVECs were cultured in chamber slides on CM for 24 h and fixed in 4% paraformaldehyde and 0.2% glutaraldedyde in phosphate-buffered saline for 1 h. Immunocytochemical analysis was performed as described in Materials and methods. Isotype control (Neg.; inset). Slides were mounted and photographed. (c) Equal amounts of proteins were used for the extraction of HUVEC membrane fractions and were subjected to immunoblot analysis for uPAR expression using specific antibodies. The blot was re-probed for DDK-tag expression.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Membrane, Cell Culture, Labeling, FACS, Expressing, Control, Saline, Extraction, Western Blot

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 3. s-uPAR colocalizes in lipid rafts on HUVECs. Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. (a) HUVECs were cultured in chamber slides on CM for 24 h and incubated with anti-uPAR antibody followed by Alexa Fluor-488- conjugated secondary antibody at 4 1C. Cells were again labeled with Alexa Fluor-595-CTxB subunit. Slides were mounted and analyzed by confocal microscopy. Negative controls, using an isotype antibody, showed no staining (inset). Serum-free medium (SFM) and DDK-tag containing rh-uPAR were used as controls. To disrupt lipid rafts, HUVECs were pretreated with MBCD, as described in Materials and methods. (b) HUVECs lipid rafts were isolated as described in Materials and methods. Lipid raft-enriched fractions were analyzed for uPAR and DDK-tag levels using immunoblot analysis. Flotillin-1 and caveolin-1 served as controls. Protein band intensities were quantified by densitometric analysis using ImageJ software (NIH). The levels of uPAR protein were normalized to protein levels in HUVECs cultured on parental-CM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM. (c) Invasion and migration assays were performed as described in Figure 1d In vitro angiogenesis assay was performed as described in Figure 1. To deplete cholesterol, HUVECs were pretreated with MBCD as described in Materials and methods (c and d). Columns: mean; bars: s.d.; n ¼ 3; *po0.01 vs parental-CM; **po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Cell Culture, Incubation, Labeling, Confocal Microscopy, Staining, Isolation, Western Blot, Software, Migration, In Vitro, Angiogenesis Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 4. s-uPAR induces ERK/Rac1-mediated migration and tube formation in HUVECs. Conditioned medium (CM) was collected from tumor cells, as described in Materials and methods. (a) HUVECs lysates were used to perform GST-Rac1 pull-down assay. The protein complexes were subjected to immunoblot analysis to detect active Rac1. Rac1 from total cell lysates was used as a control. (b) Total cell lysates were subjected to immunoblot analysis for phospho-ERK1/2 (pERK1/2) and total ERK1/2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. HUVECs grown on rh-uPAR were used as a control. (c) HUVECs were cultured on CM alone and/or supplemented with functional blocking anti-uPAR antibody (uPAR-Ab) or isotype control (Nsp.IgG.) or MEK inhibitor (U0126) for 24 h. Cell lysates or GST-Rac1 pull- down protein complexes were subjected to immunoblot analysis to detect active Rac1, Rac1 pERK1/2 and ERK1/2. GAPDH served as a loading control. (d) HUVECs were transfected with dominant-negative mutant Rac1 (Dn-Rac1) for 24 h and cultured on UR-CM. Micrographs were captured for green fluorescent protein (GFP) expression (green) and phase contrast (gray) immediately after the addition of UR-CM (magnification 60). (e) HUVECs were transfected with Dn-Rac1 for 24 h, cultured on CM for another 24 h, collected and lysed. GST-Rac1 pull- down protein complexes were subjected to immunoblot analysis to detect active Rac1. GFP and Rac1 from total cell lysates were used as controls. (f) HUVECs were transfected with Dn-Rac1 for 24 h and cultured on CM alone and/or supplemented with uPAR-Ab., or Nsp.IgG or U0126 for another 24 h. Invasion and migration assays were performed as described in Figure 1. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **Po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Migration, Pull Down Assay, Western Blot, Control, Cell Culture, Functional Assay, Blocking Assay, Transfection, Dominant Negative Mutation, Expressing

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 5. Diverse forms of tumor-associated s-uPAR in vitro and in vivo. (a) Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. CM was subjected to deglycosylation using a deglycosylation kit and analyzed by immunoblot for uPAR using specific antibodies. (b) Equal amount of proteins containing HUVEC lysates were used for extraction of cell membrane fractions and were subjected to deglycosylation, and analyzed by immunoblot for uPAR using specific antibodies. (c) In vivo angiogenic assay was performed by using the dorsal air sac model. 4910EV (EV), 4910UR (UR), 4910UR-Si (UR-Si) cells or a recombinant human uPAR (rh-uPAR) containing chamber was implanted in the dorsal cavity of mice. The micrographs for the presence of tumor-induced neovasculature (microvessels with curved thin structures and many tiny bleeding spots) and pre-existing vasculature (straight) were captured. Representative micrographs are shown. (d, e) Blood was collected from mice orthotopically xenografted with stably expressing EV, UR and UR-Si cells. Total uPAR levels were estimated using a commercial human uPAR Quantikine Immunoassay kit according to the manufacturer’s instructions. The data quantification for a set I (n ¼ 4; d) and set II (n ¼ 6; e), on day 15 and 40, respectively, after cell implantation are shown. Columns: mean; bars: s.d.; *Po0.01 vs parental control. (f) Blood serum (from mice 1–6; on day 40) was subjected to deglycosylation and analyzed by immunoblot for uPAR using specific antibodies. D2-D3, D2-D3 domain containing truncated s-uPAR; D3, D3 domain containing truncated s-uPAR; FL, full-length s-uPAR; .

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Western Blot, Extraction, Membrane, Recombinant, Stable Transfection, Expressing, Control

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 6. uPAR overexpression enhances tumor growth, vascularity and s-uPAR recruits onto endothelial cells in vivo. (a) Stably expressing EV, UR and UR-Si cells were injected intracerebrally into mice. Mice were euthanized and brains were collected and fixed as described in Materials and methods. Brain sections were stained with hematoxylin and eosin (H&E) solution, and representative micrographs are shown (upper panel). H&E-stained micrographs showing the tumor invasive front ( 20; lower panel). (b) Brain tumor areas were calculated using Image Pro Discovery Program software (Media Cybernetics, Inc., Rockville, MD, USA). Columns: mean; bars: s.d.; n ¼ 6; *Po0.01 vs parental controls. (c) Immunohistochemical analysis of brain sections using anti-uPAR and anti-vascular endothelial growth factor (VEGF). Blood vessels in tumor sections were visualized with biotin-labeled tomato lectin. Inset: isotype control. (d, e) Fluorescence microscopy for colocalization of an endothelial cell marker (von Willebrand factor (vWF)/anti-CD31) and DDK-tag in tumor sections from mice that were implanted with 4910 EV (EV) and 4910UR (UR) cells. Inset, isotype control.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Injection, Staining, Software, Immunohistochemical staining, Labeling, Control, Fluorescence, Microscopy, Marker

Journal: American Journal of Physiology-Regulatory, Integrative and Comparative Physiology

Article Title: Decreased hematopoiesis in bone marrow of mice with congestive heart failure

doi: 10.1152/ajpregu.2002.282.1.r166

Figure Lengend Snippet: Fig. 4. Increased apoptosis of CD34 hematopoietic progenitor cells in vitro by T cells (A) and natural killer (NK) cells (B). The percentage of apoptotic cells was determined with flow cytometry of cells labeled with either PI or annexin-V. Values are the means SE, n 8. *P 0.05 for mice with heart failure compared with sham-operated mice.

Article Snippet: The fraction of apoptotic CD34 cells was examined with both flow cytometric assessment of hypodiploid DNA stained with propidium iodide (19) and flow cytometric detection of surface expression of phosphatidylserine on apoptotic cells with annexin-V (Apoptosis Detection Kit, R&D Systems, Minneapolis, MN).

Techniques: In Vitro, Cytometry, Labeling

Journal: American Journal of Physiology-Regulatory, Integrative and Comparative Physiology

Article Title: Decreased hematopoiesis in bone marrow of mice with congestive heart failure

doi: 10.1152/ajpregu.2002.282.1.r166

Figure Lengend Snippet: Fig. 3. Increased apoptosis in situ of bone marrow progenitor cells in mice with heart failure. The percentage of apoptotic cells was deter- mined with flow cytometry of cells labeled with either propidium iodide (PI) or annexin-V. Values are the means SE, n 8. *P 0.05 for mice with heart failure compared with sham-operated mice.

Article Snippet: The fraction of apoptotic CD34 cells was examined with both flow cytometric assessment of hypodiploid DNA stained with propidium iodide (19) and flow cytometric detection of surface expression of phosphatidylserine on apoptotic cells with annexin-V (Apoptosis Detection Kit, R&D Systems, Minneapolis, MN).

Techniques: In Situ, Cytometry, Labeling

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) Representative flow cytometry graphs of CD11b and GFP in peripheral blood of KDRGFP mice bearing gliomas. Astrocytic tumors were generated by RCAS/TVA-mediated overexpression of PDGF as described in Methods. Low-grade gliomas were observed by week 5 and high grade by weeks 7–8. Controls (CTL) were mice bearing an intact Kdr locus without GFP knockin. (B) Quantification of CD11b+KDRGFP+ cell frequency in peripheral blood of RCAS/TVA tumor mice at low-grade and high-grade stages. ***P < 0.001, Student’s t test. LEU, Leukocytes. (C) Further characterization of murine CD11b+KDRGFP+ cells in peripheral blood by Ly6C and Ly6G staining. (D) Quantification of Ly6G+ and Ly6C+ frequency out of total CD11b+ cells in the CD11b+KDRGFP+ and CD11b+KDRGFP– populations in peripheral blood at high-grade stage. **P < 0.01, for Ly6Chi cells, Student’s t test. (E) CFU assays were performed on Lin–KDRGFP+ hematopoietic cells from BM, and macrophage (M) colonies and macrophage/granulocyte (GM) colonies, but not granulocyte (G) colonies, were observed. Quantification of various colony types formed from lineage-negative KDRGFP– or KDRGFP+ cells. ***P < 0.001, in granulocytes, Student’s t test. (F) Gene expression (quantitative RT-PCR) of various markers, including Kdr in in vitro–cultured BM Lin– cells exposed to GL261-conditioned medium at different time points. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA. Data are shown as mean ± SD.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Flow Cytometry, Generated, Over Expression, Knock-In, Staining, Gene Expression, Quantitative RT-PCR, In Vitro, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: Lethal dose–irradiated C57BL/6 mice were transplanted with Ubc-GFP Rosa26-CreERT2Kdrfl/fl and Rosa26-CreERT2KDRfl/+ BM cells, and GL261 tumors were implanted after BM engraftment. (A) Peripheral white blood cells were analyzed on side scatter (SSC) and GFP by flow cytometry. The GFP+ and GFP– populations were gated for further analysis. CD11b versus GFP (B), Ly6C versus Ly6G (C), and CD3 versus B220 (D) are shown on both GFP+ and GFP– populations. (E) BM cells were also analyzed on side scatter and GFP. (F) Lineage-negative cells were gated for analysis of HSCs and HPCs by c-Kit versus Sca-1 (G). (H) HPCs (Lin–c-Kit+Sca-1–) were further characterized by FcγR versus CD34 within both GFP+ and GFP– populations. The experiment was conducted 5 times.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Irradiation, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) The unsupervised principal component analysis of significantly altered genes in Lin–c-Kit+Sca-1– from naive mice, Lin–c-Kit+Sca-1– KDR-GFP+ cells, and Lin–c-Kit+Sca-1–KDR-GFP– cells from BM of tumor-bearing mice. (B) Lin–c-Kit+Sca-1– cells from naive mice (black), Lin–c-Kit+Sca-1– KDR-GFP+ cells (red), and Lin–c-Kit+Sca-1–KDR-GFP– cells (green) from BM of tumor-bearing mice, based on similarity of gene profiles. PC1, principal component 1. (C) The candidate genes (P < 0.05, >1.5-fold change; Lin–c-Kit+Sca-1– KDR-GFP+ versus Lin–c-Kit+Sca-1–KDR-GFP–) were divided according to the subset with the highest expression and analyzed for categories with significant enrichment (P < 0.05) of categories in GO biologic processes using DAVID tools. Similar categories were grouped accordingly. The presence of association between functions and genes was color highlighted (black [negative] versus green [positive]). (D) Expression of Id2 in various lineages of hematopoietic cells. ***P < 0.001, 1-way ANOVA. (E) Expression of ID2 in HPCs (CD45–CD34+) from patients with low-grade or high-grade gliomas. ***P < 0.001, 1-way ANOVA. n = 21. (F) Expression of ID2 in CD11b+ blood cells from patients with low-grade or high-grade gliomas. ***P < 0.001, 1-way ANOVA. n = 20. Data are shown as mean ± SD.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) Chimeric C57BL/6 mice transplanted with Id2–/– BM cells (Id2+/+ BM cells as control) were implanted with luciferase-labeled GL261 tumors intracranially. Tamoxifen was applied at day 3 after implantation. The tumors were monitored by bioluminescence. Representative images were taken at day 14. The color bar on the right represents photon intensity. The experiments had 2 replicates. The tumor growth curve is based on bioluminescence. n = 10. **P < 0.01, 1-way ANOVA. (B) Flow cytometry analysis of peripheral blood cells on CD11b, Ly6C, and Ly6G in Id2+/+ BMT and Id2–/– BMT groups. Quantification of CD11b+ cell frequency out of total white blood cells and of Ly6Chi cell frequency out of CD11b+ cells. **P < 0.01, 1-way ANOVA, for each group. n = 7. (C) Tubule formations of HCMEC/D3 (GFP) cocultured with lineage-negative Id2+/+, Id2–/–, Id2–/– scrambled sequence (SC) control, or Id2–/– KDR overexpression (OE) HPCs pretreated with TGF-β/GM-CSF. Quantifications of tubule lengths in the indicated group. ***P < 0.0001, 1-way ANOVA. n = 6. (D) Growth factor–reduced Matrigel plugs of lineage-negative Id2+/+, Id2–/–, Id2–/– SC CTL, or Id2–/– KDR overexpression HPCs pretreated with TGF-β/GM-CSF. Blood vessels (red) were perfused with rhodamine-dextran. Lower panels show magnified views to highlight vascular permeability. Quantifications of based blood vessels density (BVD) in each group. **P < 0.01, 1-way ANOVA. n = 6. Data are represented as mean ± SD. Scale bars: 50 μm (D, upper panels); 20 μm (C, D, lower panels).

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Control, Luciferase, Labeling, Flow Cytometry, Sequencing, Over Expression, Permeability

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 1. Schematic representation of experimental protocol and workflow of this present work. The top panel is the schematic illustration of the SNA substrates inserts into commercial dishes for CD34+HSPCs enrichment. Below which is the schematic representation of SMNP delivery CBE- and sgRNA-plasmids into commercial dishes with SNA substrates inserts. Then, after obtaining umbilical cord blood cells, SNA substrate and SMNP delivery mediated CBE base editing (SNA⋅SMNP⋅CBE)— enable efficiently and precisely modify BCL11A promoter to achieve C-T conversion and HBG elevation in CD34+HSPCs. The edited human CD34+HSPC was successively transplanted into SCID mouse by intraosseous injection to detect the CD34 engraftment.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Injection

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 3. supramolecular nanoparticle (SMNP) enables delivery of CBE and sgRNA plasmids to modify BCL11A to achieve HBG over expression in vitro. (A), Schematic representation of base-editing experiments in human umbilical cord blood derived CD34+HSPCs enriched by SNA substrate CBE-, sgRNA-(with a red fluorescent label (mCherry) expressing plasmid were co-delivered by SMNP in human CD34+HSPCs enriched by SNA substrate. (B), Representative images of mCherry + cells at 2 h post-transfection following SMNP delivery CBE-, sgRNA-plasmids. (C), Representative flow cytometry results of edited CD34 positive cells ratio after SMNP delivery CBE- and sgRNA- plasmids. (D), Editing efficiency of targeted BCL11A PCR products was detected by Sanger sequencing, with C-T conversion value of 32.7 % base edits at position C1, and 9.5 % at position C2.(E), Real-time PCR analysis of mCherry (left panel), BCL11A (middle panel) and HBG (right panel) expression in CD34+HSPCs subjected with/without SMNP delivering co-encapsulated CBE- and sgRNA targeting BCL11A- plasmids (edited CD34+HSPCs/ naïve CD34+HSPCs). CD34+HSPCs delivered by SMNP co-encapsulated CBE- and sgRNA targeting BCL11A- plasmids was designated as edited group, and unedited group. Results are normalized to GAPDH and shown as mean±SEM. (n=3 biological replicates). Statistical tests compare edited samples and unedited control group (**P＜ 0.01). (F), Western blots were used to detect HBG expression at protein level in edited CD34+HSPCs and naïve CD34+HSPCs. Results are representative of three biological replicates.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Over Expression, In Vitro, Derivative Assay, Expressing, Plasmid Preparation, Transfection, Flow Cytometry, Sequencing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 4. Comparison of our novel system and conventional strategy in the aspect of editing efficiency and CD34+HSPCs property. (A), Editing efficiency of targeted BCL11A PCR products from CD34+HSPCs subjected to different strategies. The top panel (SNA⋅SMNP group) is CD34+HSPCs was enriched by our SNA substrate and transfected with CBE- and sgRNA targeting BCL11A- plasmids by SMNP delivery (with C-T conversion value of 32.7 % base edits at position C1, and 9.5 % at position C2). The middle panel (IMS⋅EP group) is CD34+HSPCs was isolated by conventional immunomagnetic bead method (IMS) and transfected CBE- and sgRNA targeting BCL11A- plasmids with electroporation (EP) (with C-T conversion value of 33.7 % base edits at position C1, and 10.2 % at position C2). The bottom panel (control group) was CD34+HSPCs without edited treatment. (B), Transcriptome analysis was performed in edited CD34+HSPCs with different strategies, and the control naïve CD34+HSPCs. Venn diagram of differential expression genes among the three groups: SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs and naïve CD34+HSPCs (left panel). Gene cluster analysis of transcriptome results among the three groups: SNA⋅SMNP treated CD34+HSPCs, IMS⋅EP treated CD34+HSPCs and naïve CD34+ (right panel). (C), CD34 mRNA expression in three groups SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs and naïve CD34+HSPCs. Results shown as mean±SEM (n=3 biological replicates). (D) mRNA expression of hematopoietic stem cell marker (CD133, CD90) and cell differentiation markers (GATA-1, SPI1, CD235a and CD33) in four groups SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs, naïve CD34+HSPCs, and PMBC. CD133 and CD90 were shown in left panel, while differ entiation markers including GATA-1, SPI1, CD235a and CD33 were presented in right panel. Results shown as mean±SEM (n=3 biological replicates).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Comparison, Transfection, Isolation, Electroporation, Control, Quantitative Proteomics, Expressing, Marker, Cell Differentiation

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 5. Tracks of editing feasibility of SNA to maintain CD34 property by Intraosseous injection of transplanted edited CD34+HSPCs into SCID mouse. (A), Gene edited human CD34+HSPCs were transplanted into SCD mouse with two different injection strategies: intraosseous injection into the bone marrow of the mouse tibia (left panel) and tail vein injection (right panel). (B), Representative immunofluorescence images (scale bar=8μm) of CD34 in the bone marrow (left panel) and peripheral blood of mice at 14 weeks of intraosseous injection (top) and tail vein injection (below) strategy, respectively. (C), Representative flow cytometry results of CD45+ proportion in the bone marrow (left panel) and peripheral blood of transplanted mice with intraosseous injection (labeled with Red) and tail vein injection (labeled with green) strategy, respectively. Detection time was set at 10-, 12-, 14-week after transplantation. (D), Real-time PCR analysis of CD34 mRNA expression in the bone marrow (left panel) and peripheral blood of mice with intraosseous injection (labeled with Red) and tail vein injection (labeled with green) strategy, respectively. Detection time was set at 10-, 12-, 14-week after transplantation. Results shown as mean±SEM (n=3 biological replicates, *P<0.05, **P<0.01).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Injection, Immunofluorescence, Flow Cytometry, Labeling, Transplantation Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 6. Detection of BCL11A gene modification and HBG/HBB expression after transplantation of edited human CD34+HSPCs. (A), Representative Sanger sequencing of BCL11A from bone marrow (left panel) and peripheral blood (right panel) of transplanted mice, evidenced from C-T conversions. (B), (C), Real-time PCR was used to detect human HBG and HBB expression in the bone marrow (B) and peripheral blood (C) of SCID mice at 10, 12, 14weeks after transplantation of edited human CD34+HSPCs.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Modification, Expressing, Transplantation Assay, Sequencing, Real-time Polymerase Chain Reaction

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 7. Comparison of our novel strategy and conventional strategy in maintain CD34 property in vivo. (A), Schematic illustration of protocol. The top panel is the process of conventional strategy IMS⋅EP⋅CBE3⋅intravenous (tail vein) injection, while the below panel is the process of our novel strategy SNA⋅SMNP⋅CBE3 ⋅intraosseous injection. After transplantation edited human CD34+HSPCs with different treatment at 14 weeks, SCID mice from the two different groups were euthanatized, mononuclear cells were resorted and enriched from the bone marrow of mice in both groups. Then, transcriptome analysis was performed to detect and compare the gene expression profiles in each group with naïve CD34 controls. (B), Representative flow cytometry results of CD45+ proportion in the bone marrow of SNA⋅SMNP⋅CBE3⋅intraosseous injection mice and IMS⋅EP⋅CBE3⋅intravenous (tail vein) injection counterparts. (C), Real-time PCR analysis of human HBG expression in the bone marrow (left panel) and peripheral blood (right panel) of SNA⋅SMNP⋅CBE3⋅intraosseous injection mice and IMS⋅EP⋅CBE3⋅ intravenous (tail vein) injection counterparts. (D), Transcriptome analysis was performed in edited CD34+HSPCs with different strategies, and the control naïve CD34+HSPCs. Venn diagram of differential expression genes among the three groups: IMS⋅EP⋅CBE3⋅intravenous, SNA⋅SMNP⋅CBE3⋅intraosseous injection and naïve CD34+HSPCs (left panel). Gene cluster analysis of transcriptome results among the three groups (right panel). (E), Representative CD34 expression by Real-time PCR in the three groups: SNA⋅SMNP⋅CBE3⋅intraosseous group, IMS⋅EP⋅CBE3⋅tail vein injection counterparts, as well as naïve CD34+HSPCs. Results shown as mean±SEM (n=3 biological replicates, **P<0.01).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Comparison, In Vivo, Injection, Transplantation Assay, Gene Expression, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, Control, Quantitative Proteomics

Journal: Pathogens

Article Title: Neutrophils Expressing Programmed Death-Ligand 1 Play an Indispensable Role in Effective Bacterial Elimination and Resolving Inflammation in Methicillin-Resistant Staphylococcus aureus Infection

doi: 10.3390/pathogens13050401

Figure Lengend Snippet: PD-L1 expression is increased in the neutrophils of MRSA-infected mice. The mice were infected to MRSA by i.v. injection, and the PD-L1 expression in neutrophils was analyzed in the PB, liver, lung, spleen, and BM at 48 h post-infection. Representative plots ( A ) and cumulative percentages of PD-L1 + neutrophils ( B ) are shown. The cumulative data are shown as the mean ± standard error (SD) of six samples. Student’s t -test was used to analyze the data for significant differences. Values of * p < 0.001 are regarded as significant.

Article Snippet: Neutrophils were enriched from BM-isolated cells using the Neutrophil Isolation Kit, mouse (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany).

Techniques: Expressing, Infection, Injection

Journal: Pathogens

Article Title: Neutrophils Expressing Programmed Death-Ligand 1 Play an Indispensable Role in Effective Bacterial Elimination and Resolving Inflammation in Methicillin-Resistant Staphylococcus aureus Infection

doi: 10.3390/pathogens13050401

Figure Lengend Snippet: PD-L1 + neutrophils have enhanced anti-bacterial activity. ( A – E ) In vivo functional assay of neutrophils following PD-L1 expression. The activities were measured in the liver neutrophils 48 h after MRSA challenge. The productions of ROS ( A ), IL-1β ( B ), and MPO ( C ), and neutrophil elastase ( D ), and NET formation ( E ) were measured by flow cytometry. ( F – K ) In vitro functional assay of neutrophils. ( F ) The diagram of in vitro PD-L1 induction in neutrophils and sorting of PD-L1 + and PD-L1 − neutrophils. ( G ) Phagocytic activity against S. aureus (FITC-labeled). ROS ( H ) and IL-1β production ( I ) in HK-SA (MOI = 1:50)-stimulated neutrophils. ( J ) Cell-free-DNA (CFD) levels measured in the cultured medium of HK-SA (MOI—1:50)-stimulated neutrophils. ( K ) In vitro MRSA killing assay. Survival intracellular MRSA was quantified by measuring the CFU. The cumulative data are shown as the mean ± SD of six samples. Student’s t -test was used to analyze the data for significant differences. Values of * p < 0.05, ** p < 0.01, and *** p < 0.001 are regarded as significant.

Article Snippet: Neutrophils were enriched from BM-isolated cells using the Neutrophil Isolation Kit, mouse (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany).

Techniques: Activity Assay, In Vivo, Functional Assay, Expressing, Flow Cytometry, In Vitro, Labeling, Cell Culture

Journal: Pathogens

Article Title: Neutrophils Expressing Programmed Death-Ligand 1 Play an Indispensable Role in Effective Bacterial Elimination and Resolving Inflammation in Methicillin-Resistant Staphylococcus aureus Infection

doi: 10.3390/pathogens13050401

Figure Lengend Snippet: Bacterial structural components trigger PD-L1 expression in neutrophils through extracellular TLRs. ( A ) A diagram of the purification procedure of the MRSA structural components. ( B,C ) In vitro neutrophil stimulation. Neutrophils were isolated from WT mice BM and subjected to stimulation assay to measure the PD-L1 upregulation. ( B ) The percentages of PD-L1 + cells in the neutrophils cultured with vehicle (PBS) or HK-SA. ( C ) The percentages of PD-L1 + cells in the neutrophils cultured with vehicle (PBS), CWE, CPE, LP, DNA, or RNA isolated from MRSA. The mRNA expressions of TLR2 ( D ), TLR4 ( E ), and MyD88 ( F ) in vehicle (PBS) or HK-C60 (MOI = 1:50) cultured neutrophils. The percentages of PD-L1 + neutrophils cultured with HK-SA (MOI = 1:50) ( G ), CWE (10 μg/mL) ( H ), or LP (1 μg/mL) ( I ). The neutrophils were isolated from WT, TLR2-KO, TLR4-KO, or MyD88-KO mice. The cumulative data are shown as the mean ± SD of six samples. One-way ANOVA was used to analyze the data for significant differences. Values of * p < 0.05, ** p < 0.01, and *** p < 0.001 are regarded as significant.

Article Snippet: Neutrophils were enriched from BM-isolated cells using the Neutrophil Isolation Kit, mouse (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany).

Techniques: Expressing, Purification, In Vitro, Isolation, Cell Culture

Journal: Pathogens

Article Title: Neutrophils Expressing Programmed Death-Ligand 1 Play an Indispensable Role in Effective Bacterial Elimination and Resolving Inflammation in Methicillin-Resistant Staphylococcus aureus Infection

doi: 10.3390/pathogens13050401

Figure Lengend Snippet: PD-L1 blockade does not compromise neutrophil anti-bacterial immunity; however, it delays the resolution of inflammation in MRSA-infected mice. ( A ) Experimental design of MRSA challenge and anti-PD-L1 mAb administration. Mice were infected with MRSA and received isotype or anti-PD-L1 mAb administration. The bacterial colonization and immune functions of neutrophils were analyzed in the mice at 48 h post-MRSA challenge. ( B ) MRSA colonization in the PB, liver, lung, and spleen in MRSA-challenged mice. The CFUs are indicated as per mL for the PB and per 100 mg for the organs. ( C – E ) In vivo neutrophil functional assay. MPO ( C ) and neutrophil elastase ( D ) productions and percentage of NETosis cells ( E ) are shown. ( F ) Plasma ALT concentration in the MRSA-challenged mice. The IL-6 ( G ), TNF-α ( H ), and IL-1β ( I ) production of liver macrophages in the MRSA-challenged mice on day 7. ( J ) Liver leukocyte functional profile of the MRSA-challenged mice. ( K , L ) In vitro T cell suppression assay. T cells and neutrophils (PD-L1 − or PD-L1 + ) were co-cultured with or without isotype Ab or anti-PD-L1 mAb, and the IFN-γ producing populations of CD4 + ( K ) and CD8 + ( L ) T cells were analyzed, respectively. The cumulative data are shown as the mean ± standard error (SD) of six samples. Student’s t -test was used to analyze the data for significant differences. Values of * p < 0.05 and ** p < 0.01 are regarded as significant. ns = not significant.

Article Snippet: Neutrophils were enriched from BM-isolated cells using the Neutrophil Isolation Kit, mouse (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany).

Techniques: Infection, In Vivo, Functional Assay, Clinical Proteomics, Concentration Assay, In Vitro, Suppression Assay, Cell Culture

Journal: Pathogens

Article Title: Neutrophils Expressing Programmed Death-Ligand 1 Play an Indispensable Role in Effective Bacterial Elimination and Resolving Inflammation in Methicillin-Resistant Staphylococcus aureus Infection

doi: 10.3390/pathogens13050401

Figure Lengend Snippet: Hypothetical diagram of PD-L1 + neutrophil function in the acute phase of MRSA infection.

Article Snippet: Neutrophils were enriched from BM-isolated cells using the Neutrophil Isolation Kit, mouse (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany).

Techniques: Infection