Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways
Figure Lengend Snippet: Activation of the mitochondrial apoptosis pathway in HCMV-infected endothelial cells. A, Increased cleavage of caspases-3 and 9 in HCMV-infected endothelial cells. Total protein from mock, AD169, VHL/E, and Towne strains of infected cells (day 9) was extracted and blotted with anticaspase-9 and anticaspase-3 antibodies. β-Actin was used as an internal sample control. B, Time-dependent increases of expression and cleavage of caspase-3 in HCMV-infected cells. After indicated infection time, total protein from mock-infected and VHL/E-infected cells was extracted and blotted with antibodies against total and cleaved caspase-3. C, Cleavage of caspase-3 in HCMV-infected cells. VHL/ E-Infected HAECs (day 9) were stained with anti-IE antibody-FITC (green), anti-cleaved caspase-3 antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization. D, Cleavage of caspase-9 in HCMV-infected cells. VHL/ E-infected HAECs (day 9) were stained anti-IE antibody-FITC (green), anticleaved caspase-9 antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization. E, Time-dependent increases in expression and cleavage of caspase-8 in HCMV-infected cells. After indicated infection time, total protein from mock-infected and VHL/E-infected cells was extracted and blotted with antibody against caspase-8. F, The involvement of caspases-3, -9, and -8 in HCMV-induced endothelial apoptosis. HAECs were infected with VHL/E for 8 days and treated with 10 μmol/L of caspase inhibitor for 3 days (fresh inhibitors were added daily). TUNEL-positive cells and total cells were counted in 4 fields per coverslip. Results were expressed as the mean±SEM percentage of TUNEL-positive cells over total cells. Using ANOVA, all 3 caspase inhibitors resulted in significantly less apoptosis (P<0.001); the inhibiting effect by caspase-3 or 9 inhibitors was greater than that by caspase-8 (P=0.024).
Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.
Techniques: Activation Assay, Infection, Expressing, Staining, TUNEL Assay