immunofluorescence staining for cd62p  (Bioss)

Journal: Journal of Inflammation Research

Article Title: The EZH2 Inhibitor GSK126 Alleviates Thromboinflammation in Deep Vein Thrombosis by Suppressing TLR4 Signaling via H3K27me3 Modulation

doi: 10.2147/JIR.S551388

Figure Lengend Snippet: Tlr4 mediates H3-induced upregulation of platelet adhesion markers. ( A and B ) Expression levels of platelet adhesion markers P-selectin ( A ) and integrin αIIbβ3 ( B ). H3 stimulation significantly increased the expression of both P-selectin and integrin αIIbβ3 in platelet-rich plasma (PRP) from WT mice (**** p < 0.0001 vs WT). The H3-induced upregulation was significantly attenuated in Tlr4 -deficient mice compared to WT mice (*** p < 0.001 for P-selectin; ** p < 0.01 for integrin αIIbβ3; vs WT+H3).

Article Snippet: The membranes were blocked and then incubated overnight at 4°C with primary antibodies against TLR4 (Cell Signaling Technology, 14358, 1:1000), P-selectin (Bioss, bs-0561R, 1:1000), ICAM-1 (Bioss, bs-0608R, 1:2000), phosphorylated IκBα (Proteintech, 82349-1-RR, 1:4000), H3K27me3 (Proteintech, 61018, 1:500), and β-Actin (ZSGB-Bio, TA-09, 1:2000).

Techniques: Expressing, Clinical Proteomics

Journal: Journal of Inflammation Research

Article Title: The EZH2 Inhibitor GSK126 Alleviates Thromboinflammation in Deep Vein Thrombosis by Suppressing TLR4 Signaling via H3K27me3 Modulation

doi: 10.2147/JIR.S551388

Figure Lengend Snippet: GSK126 suppresses LPS-induced inflammation in endothelial cells via TLR4 downregulation. ( A ) Cell viability assessed by CCK-8 assay. LPS stimulation slightly increased HUVEC viability compared to the control group ( p> 0.05). Neither GSK126 treatment nor cotreatment with TAK-242 significantly altered cell viability ( p> 0.05), suggesting the anti-inflammatory effects were independent of changes in cell viability. ( B and C ) ELISA quantification of IL-6 ( B ) and TNF-α ( C ) levels in culture supernatants. LPS stimulation significantly increased the levels of IL-6 and TNF-α (*** *p< 0.0001 vs control). Compared with LPS alone, GSK126 treatment significantly reduced IL-6 secretion (* *p< 0.01) and TNF-α levels (** *p< 0.001). Compared with GSK126 alone, the addition of TAK-242 further decreased IL-6 levels ( *p< 0.05); however, cotreatment with TAK-242 did not result in an additional significant reduction in TNF-α ( p> 0.05). ( D – F ) Western blot quantification of protein levels. LPS significantly upregulated TLR4 ( D ), ICAM-1 ( E ), and P-selectin ( F ) protein expression (** *p< 0.001 vs control). GSK126 treatment significantly reduced the levels of TLR4 (* *p< 0.01), ICAM-1, and P-selectin ( *p< 0.05 vs LPS). While TLR4 levels remained elevated after cotreatment ( p< 0.05 vs control), ICAM-1 and P-selectin levels were fully restored to baseline (ns vs control). ( G ) Representative immunoblots of TLR4, ICAM-1, and P-selectin, with β-actin as the loading control.

Article Snippet: The membranes were blocked and then incubated overnight at 4°C with primary antibodies against TLR4 (Cell Signaling Technology, 14358, 1:1000), P-selectin (Bioss, bs-0561R, 1:1000), ICAM-1 (Bioss, bs-0608R, 1:2000), phosphorylated IκBα (Proteintech, 82349-1-RR, 1:4000), H3K27me3 (Proteintech, 61018, 1:500), and β-Actin (ZSGB-Bio, TA-09, 1:2000).

Techniques: CCK-8 Assay, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing