immunofluorescence staining for cd62p  (Bioss)

Journal: Bioactive Materials

Article Title: Delivering LINE1 antisense oligonucleotides via endothelial targeting extracellular vesicles to ameliorate myocardial infarction-induced cardiac senescence

doi: 10.1016/j.bioactmat.2025.07.008

Figure Lengend Snippet: CD62E and CD62P have the capacity to serve as potential targets for MI treatment. (A) RT‒qPCR analysis showing the gene expression of various adhesion molecules, including CD62E, CD62P, CD54, CD106, and CD31, in cardiomyocytes, cardiac fibroblasts, and HUVECs after 24 h of OGD treatment (n = 4). (B) Western blot analysis and quantitative data showing the expression of CD62E and CD62P in cells after 24 h of OGD treatment (n = 3). (C) Representative images of immunofluorescence staining of CD62E and CD62P in HUVECs 24 h after OGD treatment (n = 5). Scale bar, 50 μm. (D) Representative images of CD62E, CD62P and α-Actinin immunofluorescence staining in ischemic ventricular tissue (n = 4). Scale bar, 100 μm. All the data are expressed as the mean ± s.d. Significant differences were determined via one-way ANOVA with Tukey's HSD multiple comparison post hoc test. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: After being cocultured for 24 h, an OGD experiment was performed for another 24 h. Next, CD62E- and CD62P-blocking antibodies (5 μg/ml, sc-137054, sc-8419, Santa Cruz Biotechnology, Santa Cruz, CA) were added to the upper chamber of the hypoxic system for 2 h and further washed with PBS to remove excess antibody.

Techniques: Gene Expression, Western Blot, Expressing, Immunofluorescence, Staining, Comparison

Journal: Bioactive Materials

Article Title: Delivering LINE1 antisense oligonucleotides via endothelial targeting extracellular vesicles to ameliorate myocardial infarction-induced cardiac senescence

doi: 10.1016/j.bioactmat.2025.07.008

Figure Lengend Snippet: In vitro assessment of the cargo transfer capacity and therapeutic potential of SBP-LINE1-EVs. (A) Experimental design for assessing the permeation effect of SBP-EVs through HUVEC monolayers. (B-D) Representative images of DiI-labeled EVs, DiI-labeled SBP-EVs and α-Actinin immunofluorescence staining of CMs in the lower chamber under normoxic conditions (B) and OGD-induced stress conditions (C) . The intensity of DiI signals in the CMs was quantified (n = 6) (D) . An anti-CD62E antibody and/or anti-CD62P antibody was added to the upper chamber of the hypoxic system for 2 h to block CD62E and/or CD62P in HUVECs after pretreatment with OGD. Scale bar, 50 μm. (E) Immunofluorescence staining of 5-FAM-labeled LINE1-ASO delivery and α-Actinin. FAM-labeled LINE1-ASO was electroporated into EVs, and FAM-LINE1-ASO-containing SBP-EVs were added to the upper chamber of the transwell and incubated for 6 h. Scale bar, 50 μm. (F) The degree of CM senescence was analyzed via β-gal staining after the internalization of SBP-LINE1-EVs, which penetrated from the upper chamber to the lower chamber of the transwell. β-gal-positive CMs under different conditions were quantified (n = 3). Scale bar, 100 μm. (G) The expression level of LINE1 in CMs was analyzed via RT‒qPCR (n = 4). (H) Representative images of β-gal-stained CMs after treatment with Scrambled ASO-packaged SBP-EVs or LINE1-ASO-packaged SBP-EVs for 24 h. Scale bar, 100 μm; n = 3. All the data are expressed as the mean ± s.d. P values were calculated via one-way ANOVA with Tukey's HSD multiple comparison post hoc test. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: After being cocultured for 24 h, an OGD experiment was performed for another 24 h. Next, CD62E- and CD62P-blocking antibodies (5 μg/ml, sc-137054, sc-8419, Santa Cruz Biotechnology, Santa Cruz, CA) were added to the upper chamber of the hypoxic system for 2 h and further washed with PBS to remove excess antibody.

Techniques: In Vitro, Labeling, Immunofluorescence, Staining, Blocking Assay, Incubation, Expressing, Comparison

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