Mrfp egfp lc3

The mRFP-eGFP-LC3 construct created by T. Yoshimori (Kimura et al., 2007 (link)) (ptfLC3; Addgene plasmid 21074) was obtained from Addgene. Cells were pooled, seeded in chamber slides and cultured for 24 h before treatment. To analyze the autophagic flux, RGC-5 cells were transfected with ptfLC3-expressing plasmid using XtremeGENE 9 (1/2 ratio) according to the manufacturer's instructions (Roche Diagnostics) for 48 h and then treated with 4 mM EMB, 4 mM EMB and 1 µM rottlerin, 100 µM CQ or DMSO for 4 h. Cells were washed in PBS and fixed in freshly prepared 4% PFA. After three washes with PBS, the cells were mounted. Autophagic flux was determined by evaluating patterns of GFP and RFP puncta under a Zeiss confocal laser-scanning microscope and quantifying the LC3 puncta (puncta/cell were counted) using ImageJ software. Results were obtained from three independent experiments with at least 100 cells analyzed.

To visualize autophagic compartments (autophagosomes and autolysosomes), cells were transfected with plasmid ptfLC3, which encodes fusion protein mRFP-EGFP-LC3 (Addgene, plasmid #21,074 [46 (link)]) for 48 h. The transfection protocol was performed with FuGENE 6 reagent (Promega Corporation, E2691) according to the manufacturer’s instructions. In experiments designed to monitor the effect of flaviviruses on autophagy, cells were transfected for 48 h and inoculated with respective viruses 12, 24 or 48 h prior to the fixation, which terminated the experiment. Autophagy modulators (when applicable) were added to the growth medium, which was used in the last step of the transfection protocol (cells were maintained in the same medium for the total duration of 48 h).

BV2 cells (Wuhan Institute of Biological Products, Wuhan, China), a murine microglial cell line, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, United States) supplemented with 10% fetal bovine serum (FBS; Gibco). Mouse neuroblastoma (NA) cells (Wuhan Institute of Biological Products) were maintained in RPMI 1640 (Gibco) containing 10% FBS. RABV strains HEP-Flury (vaccine strain) and CVS-11 (virulent strain) were obtained from the Department of Microbiology and Immunology, School of Veterinary Medicine, South China Agricultural University (Guangzhou, China). Rabbit anti-LC3B antibody (#3868) was purchased from Cell Signaling Technology (Boston, MA, United States). Rabbit anti-p62 antibody (A0682) was purchased from ABclonal (Wuhan, China). Mouse anti-NBR1 antibody (sc-130380) was purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Mouse anti-β-actin antibody (AA128) was purchased from Beyotime (Shanghai, China). Fluorescein isothiocyanate (FITC)-labeled anti-RABV-N antibodies were obtained from Fujirebio Inc. (Malvern, PA, United States). Anti-RABV-P antibody was purchased from Zhejiang Tongdian Biotechnology Co., Ltd. (Zhejiang, China). mRFP-EGFP-LC3 (#21074) was purchased from Addgene (Cambridge, MA, United States).