The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells

Li, Li; Wang, Li; Xiao, Ruijing; Zhu, Guoguo; Li, Yan; Liu, Changxuan; Yang, Ru; Tang, Zhiqing; Li, Jie; Huang, Wei; Chen, Lang; Zheng, Xiaoling; He, Yuling; Tan, Jinquan

Indirect IF (under RNAse-free conditions), experiment was carried out as described previously [25] . The monoclonal anti-CP antibody, made in-house, was used with the the secondary FITCconjugated goat anti-rabbit IgG (Sigma). The FISH experiment was carried out as described previously [25] . To detect TMV positive RNA, the specific RNA probe sequence (5 -3 ) TTAAGT-TGCAGGACCAGAGG was used; meanwhile, as a control, the unrelated RNA probe sequence (5 -3 ) AATTCAACGTCCTG-GTCTCC was used. The probes were synthesized and labelled with Cy3 by Invitrogen Corporation. Prior to hybridization with cells, the specific probe attached to the coverslips was denatured at 75 • C for 5 min. The denatured probe was dissolved in hybridization buffer [1 mg/ml of BSA (Biolabs), 2×SSC (0.30 M NaCl/0.030 M sodium citrate), 20% dextran sulfate and 200 mM VRC (vanadyl ribonucleoside complex)] and hybridized to the cells at 37 • C overnight. After four washes in 2×SSC for 10 min, the slides were mounted with 90% (v/v) glycerol. Controls including an unrelated RNA probe were prepared and used according to the steps described above.