Mrfp gfp lc3 adenovirus

Sodium fluoride (NaF, S7920), menadione (47775), 4,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI) (28718‐90‐3) and S29434 (MCE, HY‐122614) (Sigma Aldrich); anti‐NQO2 antibody (GR257660‐8) (Santa Cruze); anti‐p‐mTOR (39321), ‐LC3A/B (7074) and ‐rabbit IgG (12741) antibodies (Cell Signaling Technology, Inc.); anti‐autophagy‐related protein 5 (ATG5) (GR3195291‐10), −p62 (GR124843‐69) and ‐GAPDH antibodies (Abcam); mRFP‐GFP‐LC3 adenovirus (Hanbio Biotechnology Co., Ltd.); packaging plasmid system (GV115, Helper 2.0) (Genechem Co., Ltd.); and all other general chemicals (Sigma Aldrich) were purchased from the sources indicated.
Neuroblastoma SH‐SY5Y cells were purchased from the Conservation Genetics CAS Kunming Cell Bank, China. Sprague–Dawley (SD) rats (4 weeks old, initial weight 90–100 g) were purchased from Liaoning Changsheng Biotechnology Co., LTD, China under the license SCXK (Liao)‐2020‐0001.

Autophagic flux was tracked by mRFP–GFP–LC3 adenovirus (Hanbio, Shanghai, China). Forty-eight hours after adenovirus infection, the cells expressed LC3 protein labeled with mRFP–GFP. Yellow puncta (mRFP⁺ and GFP ⁺) correspond to the presence of autophagosomes, and red puncta (mRFP⁺ and GFP⁻) indicate autolysosomes. Red LC3 puncta accumulation was quantified to evaluate autophagic flux. After steroid starvation for 48 h in phenol red-free medium containing 10% charcoal stripped-FBS, the cells were cultured with 0, 1 or 10 nM DHT for 3 days and then imaged under a Leica DMi8 fluorescence microscope. At least 16 cells in each group were analyzed.

Cells were seeded into 6-well plates and loaded mRFP-GFP-LC3 adenovirus (Hanbio Biotechnology, China) with a MOI (multiplicity of infection) of 50. Then, cells were treated with indicated treatments as described in figure legends. Fluorescent images were obtained with a Leica TCSSP5 laser scanning confocal microscope equipped with a 40-times objective lens. Confocal microscopy images were binarized to black and white images by using ImageJ software to quantify the number of autophagosome (yellow) and autolysosome (red) puncta per cell.

Cells were plated in a 24-well plate, transfected with mRFP-GFP-LC3 adenovirus (HanBio Technology, Shanghai, China) for 4 h, incubated with ARCSP for 48 h and observed under a confocal microscope (Zeiss, LSM880). mRFP was used to label and track LC3. Attenuation of GFP expression indicated that lysosomes had fused with autophagosomes to form autophagolysosomes, and GFP was quenched due a change in the pH, at which point only red fluorescence was detected. After microscopic imaging, the red and green fluorescence images were merged; the yellow puncta were considered autophagosomes (RFP⁺GFP⁺), and the red puncta were considered autophagolysosomes (RFP⁺GFP⁻). The intensity of autophagy flux was assessed by counting the number of yellow and red puncta. ARCSP treatment increased the numbers of yellow and red puncta, indicating that it increased autophagic flux; when the number of yellow puncta but not red puncta increased in the cells or when the numbers of yellow and red puncta decreased in the cells, autophagic flux was blocked.

In vitro study, cell autophagy was determined by western blot (WB) assays and immunofluorescence (IF). WB was performed to detect the protein abundance of ATG5, ATG7, ATG16L, LC3 I/II, P53 and SQSTM1. Immunofluorescence of LC3II and beclin1was used to evaluate the level of autophagy.
In vivo study, we used mRFP-GFP-LC3 adenovirus associated virus (AAV) (32060804, HanBio-Technology; Shanghai, China) to show the kinetics of autophagic flux. AAV was intravenously injected, according to the manufacturer’s instructions. LC3 puncta were examined by fluorescence microscopy (Olympus BX61; Japan). At the same time, autophagy markers were tested by WB assays via protein extracted from renal allograft or endothelial cells.

GC cells were transfected with an mRFP-GFP-LC3 adenovirus (HanBio, Shanghai, China) to detect autophagosomes and autolysosomes. After transfection for 6 h, the cells were treated with 5-FU or a combination of MLT and 5-FU. Autophagic flux was estimated by visualizing the cells using a fluorescence microscope 32 (link),33 (link).