hy-12815a  (MedChemExpress)

Journal: Journal of Diabetes Research

Article Title: The AGEs/RAGE Axis Contributes to Myocardial Cell Death in Type 1 Diabetic Cardiomyopathy via the ROS/NF‐ κ B/NLRP3 Inflammasome Pathway

doi: 10.1155/jdr/6438008

Figure Lengend Snippet: AGEs activate the NLRP3 inflammasome in cardiomyocytes via the RAGE/ROS/NF‐ κ B signaling axis. (a–k) Primary neonatal mouse cardiomyocytes were pretreated with or without the RAGE inhibitor FPS‐ZM1 (20 μ M) or vehicle for 1 h, followed by stimulation with AGEs (200 μ g/mL) for 24 h. (a) Representative Western blots of phospho‐NF‐ κ B p65 (pp65), total NF‐ κ B p65, NLRP3, caspase‐1 P20, and N‐GSDMD. (b–e) Quantitative analysis of the protein levels of (b) pp65/NF‐ κ B p65, (c) NLRP3, (d) caspase‐1 P20, and (e) N‐GSDMD, n = 4. (f,g) Concentrations of (f) I L‐1 β and (g) IL‐18 in the cell culture supernatant were measured by ELISA, n = 4. (h,i) Intracellular ROS levels were detected using the DCFHDA fluorescent probe. (h) Representative fluorescence images. Scale bar, 50 μ m. (i) Quantitative analysis of ROS fluorescence intensity, n = 4. (j,k) ASC oligomerization was assessed by immunofluorescence. (j) Representative images showing ASC specks. Scale bar, 10 μ m. (k) Quantification of ASC fluorescence intensity, n = 4. (l–v) Cardiomyocytes were pretreated with or without the ROS scavenger NAC (10 μ M) or vehicle for 1 h, followed by stimulation with AGEs (200 μ g/mL) for 24 h. (l) Representative immunoblots of the indicated proteins. (m–p) Quantitative analysis of (m) pp65/NF‐ κ B p65, (n) NLRP3, (o) caspase‐1 P20, and (p) N‐GSDMD, n = 4. (q,r) ELISA analysis of (q) IL‐1 β and (r) IL‐18 levels in the supernatant, n = 4. (s,t) Intracellular ROS levels were measured as above. (s) Representative images. Scale bar, 50 μ m. (T) Fluorescence quantification, n = 4. (u,v) Assessment of ASC speck formation. (u) Representative images. Scale bar, 10 μ m. (v) Quantification of ASC fluorescence intensity, n = 4.

Article Snippet: FPS‐ZM1 and MCC950 (Cat#HY‐19370 and Cat#HY‐12815), STZ (Cat#HY‐13753) were obtained from MedChemExpress (Shanghai, China).

Techniques: Western Blot, Analysis, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence, Immunofluorescence

Journal: Journal of Diabetes Research

Article Title: The AGEs/RAGE Axis Contributes to Myocardial Cell Death in Type 1 Diabetic Cardiomyopathy via the ROS/NF‐ κ B/NLRP3 Inflammasome Pathway

doi: 10.1155/jdr/6438008

Figure Lengend Snippet: AGEs induce pyroptotic cell death in cardiomyocytes through the RAGE/ROS/NLRP3 pathway. Primary neonatal mouse cardiomyocytes were pretreated with or without FPS‐ZM1 (20 μ M), NAC (10 μ M), MCC950 (10 μ M), or vehicle for 1 h, followed by stimulation with AGEs (200 μ g/mL) for 24 h. (a) Representative fluorescence images of SYTOX green staining. Scale bar, 200 μ m. (b) Quantitative analysis of SYTOX‐positive cells, n = 4. (c) Representative fluorescence images of propidium iodide (PI) uptake. Scale bar, 100 μ m. (d) Quantitative analysis of PI‐positive cells, n = 4. (e) Lactate dehydrogenase (LDH) release in the cell culture supernatant was measured, n = 4.

Article Snippet: FPS‐ZM1 and MCC950 (Cat#HY‐19370 and Cat#HY‐12815), STZ (Cat#HY‐13753) were obtained from MedChemExpress (Shanghai, China).

Techniques: Fluorescence, Staining, Analysis, Cell Culture

Journal: Journal of Extracellular Vesicles

Article Title: C1qc Mediates Blood–Brain Barrier Disruption in a Mouse Model of Intracerebral Haemorrhage Through Microglia‐Derived Migrasomes

doi: 10.1002/jev2.70377

Figure Lengend Snippet: C1qc mediates the effects of migrasomes on the BBB via the Nlrp3–Caspase 1–GSDMD pathway. (A) Immunoblots revealed C1qc, Nlrp3, cleaved caspase‐1, and GSDMD‐N protein expression levels in C1qc flox/flox model mice injected with AAV–Iba1–Cre or AAV–con ( n = 5). Densitometric quantification of protein levels; the data are presented as the mean SD; ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Immunofluorescence staining for GSDMD–N and the endothelial cell marker CD31 in the perihaematomal area of C1qc flox/flox model mice injected with AAV–Iba1–Cre or AAV–con ( n = 5). Normalized quantitative measurement of GSDMD‐N fluorescence intensity relative to CD31 area. The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) Immunofluorescence staining for cleaved caspase‐1 and the endothelial cell marker CD31 in the perihaematomal area of C1qc flox/flox model mice injected with AAV–Iba1–Cre or AAV–con ( n = 5). Fluorescence signal density from cleaved caspase‐1, normalized against the CD31 area, was analysed quantitatively. The data are expressed as ± SD; *** p < 0.001, **** p < 0.0001. (D) GSDMD‐N staining of endothelial cells that were treated as described in Figure ( n = 3). Quantification of the fluorescence intensity of GSDMD‐N of endothelial cells in the vehicle + microglia‐secreting migrasome group, the hemin + microglia‐secreting migrasome group, and the hemin + siC1qc microglia‐secreting migrasome group were also quantified. Data are presented as the mean ± SD with the indicated significance, * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Cleaved caspase‐1 staining of endothelial cells that were treated as described previously in Figure ( n = 3). Quantification of the fluorescence intensity of Cleaved caspase‐1 of endothelial cells in the vehicle + microglial migrasome groups, hemin + microglial migrasome group, and hemin + siC1qc microglial migrasome group were also quantified. The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01. (F) Immunoblots showing the levels of the Nlrp3 and GAPDH proteins in endothelial cells that were treated as described in Figure ( n = 6). Densitometric quantification of protein levels. The data are presented as the mean ± SD; *** p < 0.001. (G) Claudin‐5 staining of endothelial cells that were treated with MCC950 and microglial migrasomes without C1qc ( n = 5). Quantification of the fluorescence intensity of Claudin‐5. The data are presented as the mean ± SD; ns, no significant difference; ** p < 0.01, *** p < 0.001. (H) GSDMD‐N immunofluorescence in endothelial cells treated with migrasomes, C1qc‐deficient migrasomes, or C1qc‐deficient migrasomes plus Nigericin. Quantification of the fluorescence intensity of GSDMD‐N. The data are presented as the mean ± SD; ** p < 0.01, *** p < 0.001. (I) Claudin‐5 immunofluorescence in endothelial cells treated with migrasomes, C1qc‐deficient migrasomes, or C1qc‐deficient migrasomes plus Nigericin. Quantification of the fluorescence intensity of Claudin‐5. The data are presented as the mean ± SD; * p < 0.05, *** p < 0.001. (J) Immunoblots showing the levels of the GSDMD‐N, Claudin‐5 and GAPDH proteins in endothelial cells that were treated with migrasomes at different time points ( n = 6). Densitometric quantification of protein levels. The data are presented as the mean ± SD; ns = no significant difference, ** p < 0.01, *** p < 0.001. (K) Cell viability dramatically decreased endothelial cells that were treated with migrasomes at different time points ( n = 3). Data are shown as the means ± SD with significance levels; ns = no significant difference, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Ten milligrams of the inflammasome inhibitor MCC950 (Nlrp3 inhibitor)(Selleck Chemicals, Houston, TX, USA) was dissolved in 1 mL of 1× phosphate‐buffered saline (PBS).

Techniques: Western Blot, Expressing, Injection, Immunofluorescence, Staining, Marker, Fluorescence