Mrfp gfp lc3 adenovirus

Sugar-free DMEM medium (Glucose concentration 0mmol/L, GIBCO, USA), DMEM medium (glucose concentration 25mmol/L, HyClone USA), annexin V-FITC Apoptosis Detection Kit (Beijing 4A Biotech Co., Ltd), CCK8 Kit (Wuhan Boster Biological Technology Co., Ltd), siRNA Lyophilized Powder (Shanghai GenePharma Co., Ltd), mRFP-GFP-LC3 adenovirus (Han Heng Biotechnology Co., Ltd), rabbit anti-human β-actin, LC3AB, Cleaved Caspase 3, FoxO1 and Rab7 monoclonal antibody (Cell Signaling Technology, USA), Beclin-1 Rabbit anti-human monoclonal antibody, P62 mouse anti-human monoclonal antibody, Caspase 3 rabbit anti-human monoclonal antibody (Abcam, USA), rabbit anti-human SIRT1, Ac-FoxO1 polyclonal antibody (Santa Cruz, USA), horseradish peroxidase-labeled goat anti-mouse secondary antibodies, goat anti-rabbit secondary antibodies(Pierce, USA).

Cells were inoculated in six-well plates at 2×10⁵ cells/well. Adenovirus infection can be performed when the degree of fusion reached about 50-70%. MRFP-GFP-LC3 adenovirus (Hanheng Biotechnology Co., Ltd.) infection was performed according to the operating instructions. The infected cells were collected, seeded, and used in intervention experiments (i.e., sugar deprivation and SIRT1 expression silencing). Autophagy was observed under a fluorescence microscope (fluorescence inverted phase contrast microscope, IX50, Olympus, Japan), and autophagic flux was assessed by counting the number of intracellular fluorescent spots (fluorescent spots/cell) of GFP and RFP.

HUVECs and bEnd.3 cells were seeded into 24-well plates at a density of 1.0 × 10⁵ cells/well, and mRFP-GFP-LC3 adenovirus (HanhengBio Technology, Shanghai, China) with a multiplicity of infection (MOI) value of 50 was loaded according to the manufacturer's instructions. Subsequently, the cells were subjected to the abovementioned treatments. The cells were washed with PBS three times and fixed with a mounting medium (Solarbio, Beijing, China). Fluorescent images were acquired using a confocal laser scanning microscope equipped with a 60× objective lens (LSM 880 with Airyscan; Zeiss, Dublin, CA, USA).