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