Hoechst 33258 solution

Cells were seeded in 12-well plates (2 × 10⁵ cells/well). After attachment, the cells were treated with diverse doses of AGE. After 48 h exposure to drugs, the cells were washed twice with PBS and fixed in 4% paraformaldehyde for 20 min before staining with Hoechst 33258 solution (Beyotime, Jiangsu, China). Cells were photographed by an inverted fluorescence microscope (Olympus, Tokyo, Japan).

Cell apoptosis was analyzed by Hoechst 33258 staining and Annexin V-PE/7-AAD apoptosis detection kit (BD Biosciences) as we previously described (Liu et al., 2015 (link)). Briefly, HBVSMC were seeded at a density of 2 × 10⁵/well in 2 mL serum free DMEM. Following co-culture with EMV, RNase-EMV, or PBS (vehicle) for 24 h, cell apoptosis was measured. Cells were fixed and stained with Hoechst 33258 solution according to the manufacturer's instructions (Beyotime) followed by fluorescence microscope observation in 5 independent fields assessed from each well. The average number of positive cells and total cells per field were determined. The apoptotic rate was defined as the ratio of positive cells versus total cells. For Annexin V-PE/7-AAD apoptosis detection, cells were washed with PBS, re-suspended with 100 μL 1X annexin-binding buffer, incubated with 5 μL PE-conjugated Annexin V and 5 μL 7-Aminno-actinomycin (7-AAD) for 15 min in the dark, then analyzed by flow cytometry. Cells stained with both Annexin V-PE and 7-AAD were considered to be late apoptotic HBVSMC, while cells stained only with Annexin V-PE were considered to be early apoptotic cells. Three plates per experiment were analyzed and the experiment was repeated 3 times for each group.

Fetal bovine serum (FBS) and RPMI-1640 were purchased from GIBCO (Invitrogen, Grand Island, NY, USA). Hoechst 33258 solution, JC-1 mitochondrial membrane potential detection assay kit, reactive oxygen species assay kit and cell cycle detection kit were supplied by Beyotime Institute of Biotechnology (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was obtained from Nanjing KeyGEN Biotech. Co., Ltd. (Nanjing, Jiangsu, China). The enhanced chemiluminescence (ECL) was provided by Pierce (Thermo Scientific, New Hampshire, USA). The antibodies against P53, Bax, Bcl-2, VEGF, VEGFR, GADPH and β-actin were purchased from Wuhan Boster Biological Technology, Co., Ltd. (Wuhan, Hubei, China). Antibodies against PARP, cleaved-PARP, Caspase-3, cleaved- Caspase-3, Caspase-9 and cleaved-Caspase-9 were obtained from Cell Signaling Technology (Beverly, MA, USA). Transwell inserts with 8 μm pores polycarbonate filters were provided by Corning company (Corning Costar, Cambridge, MA, USA). Matrigel was the product of BD company (Becton Dickinson, Bedford, MA, USA).