Rapamycin rapa

Rapamycin (RAPA, #9904, Cell Signaling Technology, Danvers, MA, USA) and Torin-1 (#14379, Cell Signaling Technology) inhibitors were resuspended in DMSO, diluted in complete RPMI, and for all treatments added to cell suspensions at a final concentration of 100 nM^{24 (link)}.

All reagents we used were commercially available. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen (Carlsbad, California). Recombinant human bFGF was purchased from Sigma (Sigma‐Aldrich, St. Louis, Missouri). Anti‐GFAP, anti‐bFGF, anti‐p62, anti‐NeuN, and anti‐GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California). Anti‐GAP43, anti‐LC3, anti‐Beclin‐1, and anti‐Nestin antibodies were purchased from Abcam (CB, United Kingdom). Goat anti‐rabbit and anti‐mouse IgG‐HRP, goat anti‐chicken IgY H&L, donkey anti‐goat IgG H&L were purchased from Santa Cruz Biotechnology. An enhanced chemiluminescence kit and CM‐DiI were purchased from Bio‐Rad (Hercules, California). Thapsigargin (TG) and 3‐methyladenine (3‐MA) were purchased from Sigma‐Aldrich. The autophagy activator rapamycin (RAPA) was purchased from Cell Signaling Technology. All other reagents were purchased from Beyotime Institute of Biotechnology (Shanghai, China) unless otherwise specified.

A recombinant plasmid pMD18-T-NS5B was our laboratory stock. Thapsigargin (TG, Abcam, ab120286) and Rapamycin (RAPA, Cell Signaling, 9904), Tauroursodeoxycholic acid (TUDCA, Millipore, 580549), 4-Phenylbutyric acid (4-PBA, Sigma, P21005), and 3-methyladenine (3-MA, Sigma, M9281) were respectively dissolved in dimethyl sulfoxide (DMSO) and sterile ultrapure water as stock solutions and further diluted with culture medium to the desired concentrations. The primary antibodies used in the study were specific for GRP78 (Santa Cruz, sc-13968), phosphor-IRE1 (S724) (Abcam, ab48187), ATF-6 (ABclonal, A0202), phospho-PERK (Thr980) (Cell Signaling, 3179), phospho-eIF2α (S51) (Bioworld, BS4787), eIF2α (Bioworld, BS3651), ATF-4 (ImmunoWay, YT1102), CHOP (Santa Cruz, sc-166682), LC3B (Cell Signaling, 2775), ATG5 (Novus Biologicals, NB110-53818), Beclin1 (Cell Signaling, 3495), SQSTM1/p62 (Cell Signaling, 39749), CSFV-E2 (MEDIAN/JBT, 9011), CSFV-Npro (kindly gift from Professor Xinglong Yu, Hunan Agricultural University, China), and Tubulin (Beyotime, AT819). Alexa Fluor 488-labeled goat anti-mouse IgG(H + L) (A0428), horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG(H + L) (A0208), and goat anti-mouse IgG(H + L) (A0216) were purchased from Beyotime Biotechnology.