Journal: Journal of Diabetes Research

Article Title: CD36 Regulates PANoptosis in Diabetic Retinopathy via the NOTCH/MAML Pathway

doi: 10.1155/jdr/9324498

Figure Lengend Snippet: NOTCH pathway modulation affects HG‐induced PANoptosis. hRMECs were treated with a NOTCH agonist (Jagged1 peptide) or inhibitor (DAPT) under HG conditions. (A) Cell viability measured by CCK‐8 assay. (B) Caspase‐3/7 activity measured by colorimetric assay. (C) IL‐1 β and IL‐18 levels were quantified by ELISA. (D) Apoptosis rate measured by flow cytometry with quantification. (E) Quantification of Western blot analysis for PANoptosis‐related proteins, including cleaved Caspase‐3, GSDMD‐N, RIPK1, Bax, RIPK3, p‐MLKL, and Bcl‐2. (F) Co‐IP analysis of PANoptosome components (ASC, Caspase‐1, NLRP3, and RIPK3). (G) qPCR analysis of Bax and Bcl-2 mRNA levels. (H, I) IF staining of p‐MLKL (green) with DAPI (blue) and quantification. Scale bar = 150 μ m. Data are presented as mean ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA followed by Tukey′s post hoc test. ∗ p < 0.01, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns = not significant.

Article Snippet: To modulate NOTCH signaling under HG conditions, cells were cultured in 25.0 mmol/L D‐glucose for 72 h. During the final 24 h, the HG group was supplemented with either 5 μ g/mL Jagged1 peptide (MedChemExpress, United States) to activate the pathway or 10 μ M DAPT (MedChemExpress, United States) to inhibit it.

Techniques: CCK-8 Assay, Activity Assay, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Western Blot, Co-Immunoprecipitation Assay, Staining