Journal: Frontiers in Pharmacology

Article Title: CD226 Is Required to Maintain Megakaryocytes/Platelets Homeostasis in the Treatment of Knee Osteoarthritis With Platelet-Rich Plasma in Mice

doi: 10.3389/fphar.2021.732453

Figure Lengend Snippet: Deletion of platelet CD226 disturbed the homeostasis of MKs/platelets. (A) Identification of CD226 fl/fl and CD226 fl/fl PF4-Cre mice through PCR of DNAs extracted from the tail. (B) Flow cytometry images showing CD226 expression in CD226 fl/fl and CD226 fl/fl PF4-Cre mice. (C) HE staining of BM and spleen sections. The MKs are circled; scale bar = 50 µm. (D, E) MK number and average MK area in BM and spleen sections. (F) Routine blood test results of platelets (PLT) number, plateletcrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW), platelet-large cell ratio (P-LCR), and platelet-large cell count (P-LCC). Statistical significance between groups was analyzed using independent-sample t -tests with two-tailed p value. * p < 0.05, ** p < 0.01, ns = no statistical significance.

Article Snippet: After blocking, membranes were incubated with primary antibodies: anti-CD226 (1:500, #50232-RP02, Sino Biological, Beijing, China), anti-Beclin1 (1:500, #sc-48341, Santa Cruz Biotechnology, Heidelberg, Germany), anti-LC3 (1:500, #sc-398822, Santa Cruz Biotechnology), anti-S6 ribosomal (1:1,000, #2217, CST), anti-PDGF-A (1:500, #sc-9974, Santa Cruz Biotechnology), anti-PDGF-B (1:500, #sc-365805, Santa Cruz Biotechnology), and anti-β-actin (1:3,000, #T0022, Affinity, United States) overnight at 4 °C, and subsequently incubated with corresponding secondary antibodies for 1 hour at RT.

Techniques: Flow Cytometry, Expressing, Staining, Cell Counting, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: CD226 Is Required to Maintain Megakaryocytes/Platelets Homeostasis in the Treatment of Knee Osteoarthritis With Platelet-Rich Plasma in Mice

doi: 10.3389/fphar.2021.732453

Figure Lengend Snippet: Absence of CD226 in platelets hindered platelet maturation and reduces α-granule secretion. (A) Platelet number was evaluated by flow cytometry. (B) Percentage of TO positive and negative platelets was measured to show the maturation of platelets. Two-ANOVA was adopted for statistical analysis over time. (C) Morphological changes of platelets. Red arrow: α‐granule; blue arrow: dense-granule. (D) Area of platelet, area of total α‐granule per platelet, and average area of total α‐granule per platelet were calculated. (E) Concentrations of PDGF-AB and PF4 in serum. Statistical significance was analyzed by independent-sample t -tests with two-tailed p value. * p < 0.05, *** p < 0.001, ns = no statistical significance.

Article Snippet: After blocking, membranes were incubated with primary antibodies: anti-CD226 (1:500, #50232-RP02, Sino Biological, Beijing, China), anti-Beclin1 (1:500, #sc-48341, Santa Cruz Biotechnology, Heidelberg, Germany), anti-LC3 (1:500, #sc-398822, Santa Cruz Biotechnology), anti-S6 ribosomal (1:1,000, #2217, CST), anti-PDGF-A (1:500, #sc-9974, Santa Cruz Biotechnology), anti-PDGF-B (1:500, #sc-365805, Santa Cruz Biotechnology), and anti-β-actin (1:3,000, #T0022, Affinity, United States) overnight at 4 °C, and subsequently incubated with corresponding secondary antibodies for 1 hour at RT.

Techniques: Flow Cytometry, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: CD226 Is Required to Maintain Megakaryocytes/Platelets Homeostasis in the Treatment of Knee Osteoarthritis With Platelet-Rich Plasma in Mice

doi: 10.3389/fphar.2021.732453

Figure Lengend Snippet: Platelet-specific CD226 absence led to abnormal ribosomal function and structure. (A) A GO analysis of the DEPs. (B) Annotation of the gene ID. (C) Volcano plot of differentially expressing proteins from the CD226 fl/fl PF4-Cre platelet versus littermates ( n = 3 per group).

Article Snippet: After blocking, membranes were incubated with primary antibodies: anti-CD226 (1:500, #50232-RP02, Sino Biological, Beijing, China), anti-Beclin1 (1:500, #sc-48341, Santa Cruz Biotechnology, Heidelberg, Germany), anti-LC3 (1:500, #sc-398822, Santa Cruz Biotechnology), anti-S6 ribosomal (1:1,000, #2217, CST), anti-PDGF-A (1:500, #sc-9974, Santa Cruz Biotechnology), anti-PDGF-B (1:500, #sc-365805, Santa Cruz Biotechnology), and anti-β-actin (1:3,000, #T0022, Affinity, United States) overnight at 4 °C, and subsequently incubated with corresponding secondary antibodies for 1 hour at RT.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: CD226 Is Required to Maintain Megakaryocytes/Platelets Homeostasis in the Treatment of Knee Osteoarthritis With Platelet-Rich Plasma in Mice

doi: 10.3389/fphar.2021.732453

Figure Lengend Snippet: CD226 deficiency in platelets decreased the ribosome and autophagy related protein expression in MK/platelets and human Dami cells. (A) S6 ribosomal protein-expressing ribosome in isolated MKs from BM. Scale bar = 50 µm. (B) Lysates of freshly isolated mice platelets were subjected to western blot with anti-S6 ribosomal, PDGF-A, and PDGF-B antibodies. (C) Double immunofluorescence staining for vWF and Beclin1 in the BM slides from CD226 fl/fl PF4-Cre and CD226 fl/fl mice. Scale bar = 50 µm. (D) Lysates of freshly isolated mice platelets were subjected to western blot with anti-LC3, Beclin1, and CD226 antibodies. (E) Dami cells infected with lentivirus shCD226 were subjected to western blot with anti-LC3, Beclin 1, and CD226 antibodies.

Article Snippet: After blocking, membranes were incubated with primary antibodies: anti-CD226 (1:500, #50232-RP02, Sino Biological, Beijing, China), anti-Beclin1 (1:500, #sc-48341, Santa Cruz Biotechnology, Heidelberg, Germany), anti-LC3 (1:500, #sc-398822, Santa Cruz Biotechnology), anti-S6 ribosomal (1:1,000, #2217, CST), anti-PDGF-A (1:500, #sc-9974, Santa Cruz Biotechnology), anti-PDGF-B (1:500, #sc-365805, Santa Cruz Biotechnology), and anti-β-actin (1:3,000, #T0022, Affinity, United States) overnight at 4 °C, and subsequently incubated with corresponding secondary antibodies for 1 hour at RT.

Techniques: Expressing, Isolation, Western Blot, Double Immunofluorescence Staining, Infection

Journal: Frontiers in Pharmacology

Article Title: CD226 Is Required to Maintain Megakaryocytes/Platelets Homeostasis in the Treatment of Knee Osteoarthritis With Platelet-Rich Plasma in Mice

doi: 10.3389/fphar.2021.732453

Figure Lengend Snippet: Deletion of platelet CD226 diminished the protective effects of PRP on articular cartilage of DMM mice, and PDGF restored it. (A) HE and safranin O-fast green staining of sagittal sections of the medial compartment of the tibia. Scale bar = 100 µm.

Article Snippet: After blocking, membranes were incubated with primary antibodies: anti-CD226 (1:500, #50232-RP02, Sino Biological, Beijing, China), anti-Beclin1 (1:500, #sc-48341, Santa Cruz Biotechnology, Heidelberg, Germany), anti-LC3 (1:500, #sc-398822, Santa Cruz Biotechnology), anti-S6 ribosomal (1:1,000, #2217, CST), anti-PDGF-A (1:500, #sc-9974, Santa Cruz Biotechnology), anti-PDGF-B (1:500, #sc-365805, Santa Cruz Biotechnology), and anti-β-actin (1:3,000, #T0022, Affinity, United States) overnight at 4 °C, and subsequently incubated with corresponding secondary antibodies for 1 hour at RT.

Techniques: Staining

Journal: Frontiers in Pharmacology

Article Title: CD226 Is Required to Maintain Megakaryocytes/Platelets Homeostasis in the Treatment of Knee Osteoarthritis With Platelet-Rich Plasma in Mice

doi: 10.3389/fphar.2021.732453

Figure Lengend Snippet: Histological scoring of PRP-treated DMM mice. (A) OARIS-modified Mankin score of articular cartilage. (B) Osteophyte size scores and (C) Osteophyte maturity scores. The score-related data were analyzed by Kruskal–Wallis test accompanied with Dunn’s multiple comparison test. * p < 0.05, ** p < 0.01, ns = no statistical significance. . Flow cytometry analysis for the expression of CD226 in splenocyte from CD226 fl/fl and CD226 fl/fl PF4-Cre mice. (A) Gating strategy for flow cytometric analysis of splenocyte subsets. (B) Representative image of CD226 expression levels in B cells, T cells, NK cells, CD11b + myeloid cells and CD11c + DC. . Other parameter detected after platelet CD226 deficiency in mice. (A) Routine blood test of the hemoglobin (HGB), red blood cell (RBC) number, and white blood cell (WBC) number. (B) Area of total dense-granule per platelet and average area of dense-granule per platelet in two groups. Statistical significance between groups was analyzed using independent-sample t -tests with two-tailed p value. ns = no statistical significance.

Article Snippet: After blocking, membranes were incubated with primary antibodies: anti-CD226 (1:500, #50232-RP02, Sino Biological, Beijing, China), anti-Beclin1 (1:500, #sc-48341, Santa Cruz Biotechnology, Heidelberg, Germany), anti-LC3 (1:500, #sc-398822, Santa Cruz Biotechnology), anti-S6 ribosomal (1:1,000, #2217, CST), anti-PDGF-A (1:500, #sc-9974, Santa Cruz Biotechnology), anti-PDGF-B (1:500, #sc-365805, Santa Cruz Biotechnology), and anti-β-actin (1:3,000, #T0022, Affinity, United States) overnight at 4 °C, and subsequently incubated with corresponding secondary antibodies for 1 hour at RT.

Techniques: Modification, Flow Cytometry, Expressing, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy

Article Title: Chemotherapy-resistant osteosarcoma is highly susceptible to IL-15-activated allogeneic and autologous NK cells

doi: 10.1007/s00262-010-0965-3

Figure Lengend Snippet: a Examples of immunohistochemical staining of ligands for the activating receptors NKG2D (MICA) and DNAM-1 (CD112 and CD155) and of β2-microglobulin, HLA-A (HCA2), and HLA-B/C (HC10) on osteosarcoma samples. b Overview of the results of immunohistochemical stainings on pre-chemotherapy and post-chemotherapy samples of the primary tumor as well as metastatic osteosarcoma tissue. Expression levels of CD112 and CD155 but not the other ligands decreased significantly upon chemotherapy treatment ( P -value Kruskal–Wallis test <0.001 as ***). c Example of flow cytometry plots for MICA, CD112, CD155, and HLA class I for the osteosarcoma cell line IOR/OS-14; isotype-matched control staining is shown in gray

Article Snippet: For blocking experiments, NK cells were pre-incubated with blocking anti-NKG2D (R&D systems, clone 149810) and/or blocking anti-DNAM-1 (BD Pharmingen, clone DX11) at a concentration of 20 μg/ml.

Techniques: Immunohistochemical staining, Staining, Expressing, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy

Article Title: Chemotherapy-resistant osteosarcoma is highly susceptible to IL-15-activated allogeneic and autologous NK cells

doi: 10.1007/s00262-010-0965-3

Figure Lengend Snippet: a cytolysis of U2-OS by unstimulated ( solid lines ) and IL-15-activated ( dashed lines ) NK cells was almost completely abrogated when the NK cells were pre-incubated with both anti (α)-DNAM-1- and α-NKG2D-blocking antibodies ( filled square vs. open square ). Unstimulated NK cells were most dependent on DNAM-1 ( open circle ) signaling, whereas activated NK cells were most dependent on NKG2D ( filled circle ). Error bars represent standard error of the mean lysis of experiment performed in triplicate. Similar results were obtained for SAOS-2, HOS, and ZK-58 using unstimulated ( b ) and IL-15-activated NK cells ( c ). Bars represent mean lysis in at least three independent experiments using healthy donor NK cells; error bars represent standard error of the mean. Friedman test, Dunn’s post-test compared to non-blocked; P -value <0.05 noted as *; <0.01 as **; <0.001 as ***

Article Snippet: For blocking experiments, NK cells were pre-incubated with blocking anti-NKG2D (R&D systems, clone 149810) and/or blocking anti-DNAM-1 (BD Pharmingen, clone DX11) at a concentration of 20 μg/ml.

Techniques: Incubation, Blocking Assay, Lysis

Journal: Immunotherapy Advances

Article Title: DNAM1 and TIGIT balance the T cell response, with low T cell TIGIT expression corresponding to inflammation in psoriatic disease

doi: 10.1093/immadv/ltaa004

Figure Lengend Snippet: Higher TIGIT and comparable DNAM1 expression by CD8 versus CD4 T cells. Flow cytometry analysis of CD4 (square) and CD8 (circle) T cells ex vivo of healthy controls (HC, n = 7, symbol with cross), psoriasis (PsO, n = 7, blank symbol), and psoriatic arthritis (PsA, n = 7, filled symbol) patients. (A–D) Pooled data of all subjects. (E–F) Data of HC, PsO, and PsA patients shown separately. (A) Significantly higher proportion of TIGIT-positive CD8 T cells compared to CD4 T cells. (B) Comparable proportion of DNAM1-positive CD8 and CD4 T cells. (C) Significantly higher TIGIT MFI of CD8 T cell compared to CD4 T cells. (D) Comparable DNAM1 MFI of CD4 and CD8 T cells. (E) Comparable proportion of TIGIT-positive CD4 T cells in HC, PsO, and PsA. (F) Comparable proportion of DNAM1-positive CD4 T cells in HC, PsO and PsA. (G) Comparable proportion of TIGIT-positive CD8 T cells in HC, PsO, and PsA. (G) Comparable proportion of DNAM1-positive CD8 T cells in HC, PsO, and PsA. *Significant P -value MWU.

Article Snippet: To assess to assess the effect of TIGIT and DNAM1 blockade on T cell activation and proliferation, we cultured PBMCs in complete medium (RPMI 1640 + 10% fetal bovine serum + 1% Penicillin-Streptomycin) with 10 µg/ml TIGIT blocking antibody (16-9500-82, Invitrogen), 10 µg/ml DNAM1 blocking antibody (559787, BD Pharmingen), or 10 µg/ml isotype control for TIGIT (16-4714-82, Invitrogen) and DNAM1 (555746, BD Pharmingen).

Techniques: Expressing, Flow Cytometry, Ex Vivo

Journal: Immunotherapy Advances

Article Title: DNAM1 and TIGIT balance the T cell response, with low T cell TIGIT expression corresponding to inflammation in psoriatic disease

doi: 10.1093/immadv/ltaa004

Figure Lengend Snippet: TIGIT blockade increases T cell proliferation, and DNAM1 blockade reduces T cell pro-inflammatory cytokine production. Flow cytometry analysis of PBMCs stimulated for 3 days with CD3/CD28 Dynabeads (PBMC:Dynabead 10:1), after either 10 µg/ml DNAM1 blocking antibody, 10 µg/ml TIGIT blocking antibody or 10 µg/ml DNAM1 and TIGIT blocking antibody isotypes. Pooled data of healthy controls, psoriasis, and psoriatic arthritis patients. Shown are percentages of proliferated T cells, stained with 2 µM CellTrace Violet reagent (A, B, G, and H) and percentages of TNF and IFNγ producing T cells after 4 hours re-stimulation with 50 ng/ml PMA, 1 µg/ml ionomycin in the presence of Brefeldin A (1:1000) (C–F, H–K). (A) TIGIT block significantly increases CD4 T cell proliferation. (B) DNAM1 block has no significant effect on CD4 T cell proliferation. (C) No significant difference in CD4 T cell TNF production after TIGIT blockade (44.5% vs. 45.4%, P > 0.05). (D) No significant decrease in TNF production by CD4 T cells after DNAM1 block (44.5% vs. 39.7%, P > 0.05). (E) No significant increase in CD4 T cell IFNγ production after TIGIT blockade (15.6% vs. 17.4%, P > 0.05). (F) Significantly decreased IFNγ production by CD4 T cells after DNAM1 block (15.6% vs. 13.0%, P = 0.0015). (G) TIGIT block significantly increases CD8 T cell proliferation. (H) DNAM1 block has no significant effect on CD8 T cell proliferation. (I) No significant difference in CD8 T cell TNF production after TIGIT blockade (35.8% vs. 36.1%, P > 0.05). (J) Significant decrease in TNF production by CD8 T cells after DNAM1 block (36.1% vs. 29.6%, P = 0.0039). (K) Trend toward increased CD8 T cell IFNγ production after TIGIT blockade (29.4% vs. 32.0%, P > 0.05). (L) Significant decrease in IFNγ production by CD8 T cells after DNAM1 block (29.4% vs. 25.9%, P = 0.0140). *Significant P -value Wilcoxon-signed rank test.

Article Snippet: To assess to assess the effect of TIGIT and DNAM1 blockade on T cell activation and proliferation, we cultured PBMCs in complete medium (RPMI 1640 + 10% fetal bovine serum + 1% Penicillin-Streptomycin) with 10 µg/ml TIGIT blocking antibody (16-9500-82, Invitrogen), 10 µg/ml DNAM1 blocking antibody (559787, BD Pharmingen), or 10 µg/ml isotype control for TIGIT (16-4714-82, Invitrogen) and DNAM1 (555746, BD Pharmingen).

Techniques: Flow Cytometry, Blocking Assay, Staining