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: The serum concentrations of PDGF‐AB and platelet factor 4 (PF4) were analyzed using their corresponding ELISA kits (#EK0486 for PDGF-AB, #EK0727 for PF4; Boster, Wuhan, China) according to the manufacturer’s instructions.

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: The serum concentrations of PDGF‐AB and platelet factor 4 (PF4) were analyzed using their corresponding ELISA kits (#EK0486 for PDGF-AB, #EK0727 for PF4; Boster, Wuhan, China) according to the manufacturer’s instructions.

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: The serum concentrations of PDGF‐AB and platelet factor 4 (PF4) were analyzed using their corresponding ELISA kits (#EK0486 for PDGF-AB, #EK0727 for PF4; Boster, Wuhan, China) according to the manufacturer’s instructions.

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: The serum concentrations of PDGF‐AB and platelet factor 4 (PF4) were analyzed using their corresponding ELISA kits (#EK0486 for PDGF-AB, #EK0727 for PF4; Boster, Wuhan, China) according to the manufacturer’s instructions.

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: 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: The serum concentrations of PDGF‐AB and platelet factor 4 (PF4) were analyzed using their corresponding ELISA kits (#EK0486 for PDGF-AB, #EK0727 for PF4; Boster, Wuhan, China) according to the manufacturer’s instructions.

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

Journal: Nature Communications

Article Title: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

doi: 10.1038/ncomms11996

Figure Lengend Snippet: ( a ) Human angiogenesis array analysis of the conditional medium from SW480-controland SW480-SARI cells. ( b ) Summary of the relative signal intensity of indicated cytokines. ( c ) VEGF exacted from SW480 and HCT116 cells was quantified by ELISA ( n =3; ** P <0.01; Student's t -test). ( d ) Western blot showing expression of the SARI protein and that SARI inhibits cellular VEGF expression in SW480 and HCT116 cells in vitro . ( e ) Staining for SARI (red) and VEGF (green) in SW480-control, SW480-SARI, HCT116-control and HCT116-SARI cells; DAPI staining for the nucleus. Scale bar, 50 μm. ( f , g ) Endothelial tube formation estimation following the incubation of HUVECs with conditioned medium from SW480-control, SW480-SARIcancer cells with or without shVEGF-442, or shVEGF-901 transfection. The number of branches was quantified ( g ; n =5; P <0.01; NS, no significant difference; Student's t -test). Scale bar, 50 μm. ( h , i ) Representative photomicrographs of HUVECs that have invaded through Matrigel chambers after incubation with conditioned medium for 72 h from SW480-control, SW480-SARI cancer cells with or without shVEGF-442, or shVEGF-901 transfection. Scale bar, 50 μm. ( i ) Quantification of HUVECs that have invaded through Matrigel chambers ( n =5; P <0.01; NS, no significant difference; Student's t -test). Ctrl, control.

Article Snippet: The supernatant was used to measure the total levels of several cytokines using the human VEGF ELISA kit (NeoBioscience, Shenzhen, China), PDGF-BB ELISA kit (Boster, Wuhan, China), bFGF ELISA kit (NeoBioscience), VEGF-C ELISA kit (Boster), PIGF ELISA kit (NeoBioscience) and VEGF-D ELISA kit (NeoBioscience) according to the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, In Vitro, Staining, Control, Incubation, Transfection

Journal: Nature Communications

Article Title: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

doi: 10.1038/ncomms11996

Figure Lengend Snippet: ( a ) Co-immunoprecipitation products after adding anti-SARI antibody were separated by SDS–PAGE, and the differential band was analysed by MS. ( b ) Co-immunoprecipitation analysis of SW480-control and SW480-SARI cell lysates using anti-SARI and anti-Cp antibody. ( c ) Staining for Cp (green) and SARI (red) in SW480-control and SW480-SARI cells; DAPI staining for the nucleus. Scale bar, 50 μm. ( d ) Immunoblots of SARI and Cp expression in SW480 and HCT116-blank, control and SARI cells. GAPDH was used as a loading control. ( e ) Western blotting detection of Cp expression of collected nuclear and cytoplasmic proteins. Histone H3 was used as the nucleus loading control and β-actin as the cytoplasm loading control. ( f ) Immunoblots of Cp and VEGF expression in SW480-control and SW480-SARI cells with or without MG132 treatment. GAPDH was used as the loading control. ( g ) ELISA detection of VEGF expression in SW480-control and SW480-SARI cells with or without MG132 treatment. ( n =4; ** P <0.01; Student's t -test) ( h ) Measurement of Cp in cell lysates harvested at 0, 2, 4, 6, 8 and 10 h after the addition of cycloheximide (CHX) to arrest protein synthesis. ( i ) Densitometry analysis of Cp expression in multiple western blots after CHD addition ( n =3; ** P <0.01; Student's t -test). ( j ) Immunoblots of Cp expression in HCT15, HCT15-shNC, HCT15-shSARI-1068 and HCT15-shSARI-554 cells. GAPDH was used as a loading control. ( k ) Co-immunoprecipitation analysis using anti-HA and anti-CP antibodies of total cell lysates collected from SW480 cells after transfection with HA-SARI (encoding 1–79 amino acids), HA-SARI (encoding 80–274 amino acids) and HA-SARI (encoding 1–274 amino acids). Immunoblots of HA and Cp expression. ( l ) Immunoblots of HA and Cp expression in SW480 cells after transfection with pVax, HA-SARI (encoding 1–79 amino acids), HA-SARI (encoding 80–274 amino acids) and HA-SARI (encoding 1–274 amino acids). GAPDH was used as a loading control. ( m ) ELISA detection of VEGF expression in the conditional medium from the SW480 cells after transfection with pVax, HA-SARI (encoding 1–79 amino acids), HA-SARI (encoding 80–274 amino acids) and HA-SARI (encoding 1–274 amino acids) with or without TM (4.0 μM) treatment. ( n =3; ** P <0.01; analysis of variance analysis). IB, immuoblot.

Article Snippet: The supernatant was used to measure the total levels of several cytokines using the human VEGF ELISA kit (NeoBioscience, Shenzhen, China), PDGF-BB ELISA kit (Boster, Wuhan, China), bFGF ELISA kit (NeoBioscience), VEGF-C ELISA kit (Boster), PIGF ELISA kit (NeoBioscience) and VEGF-D ELISA kit (NeoBioscience) according to the manufacturer's instructions.

Techniques: Immunoprecipitation, SDS Page, Control, Staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Nature Communications

Article Title: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

doi: 10.1038/ncomms11996

Figure Lengend Snippet: ( a ) Immunoblots of SARI and HIF-1α expression in SW480 and HCT116 cells after stable expression of SARI. HCT116-control and HCT116-SARI cells were incubated under 1% O 2 for 48 h or treated with DMOG for 24 h. β-Actin was used as a loading control. ( b ) Immunoblots of Cp, HIF-1α and VEGF expression in control and SARI cells with or without PX-478 (45 μM) or Tm (4.0 μM) treatment under normoxia and hypoxia. GAPDH was used as the loading control. ( c ) Immunoblots of Cp, HIF-1α and VEGF expression in control and SARI cells after transfection with or without p-Cp plasmid under normoxia and hypoxia. GAPDH was used as the loading control. ( d ) Expression of VEGF exacted by SW480-control and SW480-SARI cells with or without Tm (4.0 μM) and PX-478 (45 μM) treatment, after transfection with or without Cp expression plasmid ( n =3; ** P <0.01, NS, no significant difference; Student's t -test). ( e ) Endothelial tube formation estimation following the incubation of HUVECs with conditioned medium from SW480-control and SW480-SARI cells with or without Tm treatment (4.0 μM) and PX-478 (45 μM), after transfection with or without Cp expression plasmid. The number of branches were quantified ( n =5; ** P <0.01; NS, no significant difference; Student's t -test). Scale bar, 50 μm. ( f ) Representative photomicrographs of HUVECs that have invaded through Matrigel chambers after incubation with conditioned medium from SW480-control and SW480-SARI cells with or without Tm treatment (4.0 μM) and PX-478 (45 μM), after transfection with or without Cp expression plasmid. Quantification of HUVECs that have invaded through Matrigel chambers ( n =5; ** P <0.01; NS, no significant difference; Student's t -test). ( g ) Immunoblots of Cp and HIF-1α expression in HCT15, HCT15-shNC, HCT15-shSARI-1068 and HCT15-shSARI-554 cells with or without Tm (4.0 μM) treatment. GAPDH was used as a loading control. ( h ) Expression of VEGF exacted by HCT15, HCT15-shNC, HCT15-shSARI-1068 and HCT15-shSARI-554 cells with or without Tm treatment (4.0 μM) was measured by ELISA ( n =3; ** P <0.01; ## P <0.01; analysis of variance analysis).

Article Snippet: The supernatant was used to measure the total levels of several cytokines using the human VEGF ELISA kit (NeoBioscience, Shenzhen, China), PDGF-BB ELISA kit (Boster, Wuhan, China), bFGF ELISA kit (NeoBioscience), VEGF-C ELISA kit (Boster), PIGF ELISA kit (NeoBioscience) and VEGF-D ELISA kit (NeoBioscience) according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Control, Incubation, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

doi: 10.1038/ncomms11996

Figure Lengend Snippet: ( a ) Staining for VEGF (red) revealed less VEGF-positive cells in SARI than in controlSW480Matrigel plugs, Scale bar, 50 μm; VEGF-positive (%; VEGF positive per total cells; n =5; ** P <0.01; Student's t -test). ( b ) Staining for VEGF (red) revealed less VEGF-positive cells in SARI than in control HCT116 tumours ( d ), Scale bar, 100 μm; VEGF positive (%;VEGF positive per total cells; n =5; ** P <0.01; Student's t -test). ( c ) Staining for Cp revealed less Cp-positive cells in SARI than in control HCT116 tumours, Scale bar, 100 μm; Cp positive (%;Cp positive per total cells; n =5; ** P <0.01; Student's t -test). ( d ) Staining for HIF-1α (red)-positive cells in SARI and control HCT116 tumours, Scale bar, 100 μm; HIF-1α positive (%; HIF-1α positive per total cells; n =5; ** P <0.01; Student's t -test). ( e ) Immunoblots of VEGF, Cp and HIF-1a expression in SW480-control and SW480-SARI tumours. GAPDH was used as the loading control. ( f ) ELISA detection of VEGF expression in SW480-control and SW480-SARI tumours ( n =3; ** P <0.01; Student's t -test). ( g ) Staining for CD31, VEGF, Cp and HIF-1α in colonic tumours of AOM/DSS induction. Analysis of the number of microvessels, VEGF-positive cells, Cp-positive cells and HIF-1α-positive cells ( n =4; ** P <0.01; Student's t -test). Scale bar, 100 μm. ( h ) Immunoblots of VEGF, Cp and HIF-1α expression in colonic tumours of SARI −/− and SARI WT mice after AOM/DSS induction. GAPDH was used as the loading control. ( i ) ELISA detection of VEGF expression in colonic tumours of SARI −/− and SARI WT mice after AOM/DSS induction. ( n =3; ** P <0.01; Student's t -test) ( j ) Nuclear and cytoplasmic proteins were collected from the colonic tumours of SARI −/− and SARI WT mice after AOM/DSS induction. Western blotting was performed to detect Cp expression. Histone H3 was used as the nuclear loading control and β-actin as the cytoplasmic loading control.

Article Snippet: The supernatant was used to measure the total levels of several cytokines using the human VEGF ELISA kit (NeoBioscience, Shenzhen, China), PDGF-BB ELISA kit (Boster, Wuhan, China), bFGF ELISA kit (NeoBioscience), VEGF-C ELISA kit (Boster), PIGF ELISA kit (NeoBioscience) and VEGF-D ELISA kit (NeoBioscience) according to the manufacturer's instructions.

Techniques: Staining, Control, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular medicine reports

Article Title: Protective effects of glutamine in a rat model of endotoxemia.

doi: 10.3892/mmr.2012.1007

Figure Lengend Snippet: Figure 4. Immunolocalization of (A-C) platelet-derived growth factor-B (PDGF-B) and (D-F) PDGF receptor-β (PDGFR-β) in brain tissue of (A,D) controls, (B,E) lipopolysaccharide (LPS) group at 72 h, and the (C,F) glutamine (Gln) treatment group at 72 h. PDGF-B immunoreactivity was observed in the cyto plasm, PDGFR-β immunoreactivity was observed in the membrane of cortical neurons.

Article Snippet: Rabbit anti-rat NF-κB, PDGF-B, and PDGFR-β antibodies and ABC kits were purchased from Boster (Wuhan, China).

Techniques: Derivative Assay, Membrane

Journal: Molecular medicine reports

Article Title: Protective effects of glutamine in a rat model of endotoxemia.

doi: 10.3892/mmr.2012.1007

Figure Lengend Snippet: Figure 6. Western blot analysis of PDGF receptor-β (PDGFR-β) protein at various time points following injection of lipopolysaccharide (LPS). Upper row depicts the control group from left to right as 1-5 (2, 6, 12, 24 and 72 h) and the treatment group as 6-10 (2, 6, 12, 24 and 72 h). Lower row depicts the control group from left to right as 1-5 (2, 6, 12, 24 and 72 h) and the LPS group as 6-10 (2, 6, 12, 24 and 72 h).

Article Snippet: Rabbit anti-rat NF-κB, PDGF-B, and PDGFR-β antibodies and ABC kits were purchased from Boster (Wuhan, China).

Techniques: Western Blot, Injection, Control