Fig. 4

Inhibition of SIRT1 expression restrains autophagy and RV replication. (A) The mRNA expression of SIRT1 in Caco-2 sh-SIRT1 cells was detected by qRT-PCR. (B) Caco-2 cells transfected with sh-NC, sh-SIRT1#1 or sh-SIRT1#2 were infected with RV-Wa (MOI = 1) for 24 h, and the cells were then harvested for detection. The expression of RV structural proteins VP6 and VP7 were detected by western blot. (C) The expression of VP6 was detected by Immunofluorescence analysis. (D) The RV titers was detected by using plaque assays. E/F. The survival of Caco-2 cells were assessed by MTT and plate cloning. G. The expression of Cleaved caspase 3 and Caspase 3 were detected by western blot. H. The expression of autophagy-linked protein LC3 was detected by Immunofluorescence. I. The Beclin1 and SIRT1 were determined by western blot. J. Caco-2 cells transfected with oe-SIRT1 or oe-NC were infected with RV-Wa (MOI = 1), and then treated with DMEM or bafilomycin A1 (Baf-A1) for 24 h, the cells were then harvested for detection. The expressions of Beclin1, SIRT1 and VP6 were determined by western blot. K. The infectious virus titers in cell supernatants were evaluated by using plaque assays. L. The survival of Caco-2 cells were assessed by MTT. The fold change was calculated after normalized with GAPDH. The data are presented as the mean ± SD. # vs. the sh-NC, P < 0.05