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. 2017 May 17;25(8):1900–1916. doi: 10.1016/j.ymthe.2017.04.022

Figure 4.

Figure 4

Sulforaphane-Enhanced VSVΔ51 Replication and Oncolysis Is Dependent on Nrf2-Dependent Autophagy

(A) Immunofluorescence analysis of U-2 OS cells was conducted to detect changes in the levels of p62 and LC3-II in response to a 24 hr treatment with SFN (20 μM). (B) PC-3 cells were pretreated with SFN (20 μM) for 24 hr and were subsequently infected with VSVΔ51-GFP (MOI 1). Whole-cell extracts were analyzed by immunoblotting for LC3B, VSV proteins, ATG5, and β-actin. Results are from a representative experiment; all immunoblots are from the same samples. (C) PC-3 cells were pretreated with SFN (20 μM) in the presence or absence of the PI3K/autophagy inhibitor 3-MeA (10 mM) for 24 hr. Cells were subsequently infected with VSVΔ51-GFP (MOI 1). Infectivity was quantified by flow cytometry at 24 hr post-infection. Data are the means ± SEM from three independent experiments. (D–F) WT Atg5 and Atg5−/− MEFs were stimulated overnight with SFN (7.5 μM) before challenge with VSVΔ51 (MOI 0.01). Viral infectivity was determined by flow cytometry (D) and viral replication by plaque assay (E). (F) Whole-cell extracts were analyzed by immunoblotting for LC3B, VSV proteins, ATG5, and β-actin. (G and H) A549 cells were transfected with control, Nrf2, or HO-1 siRNA and 48 hr later were treated with SFN (15 μM) for an extra 24 hr. Whole-cell extracts were analyzed by immunoblotting for Nrf2, HO-1, LC3, and β-actin. (I) Immunofluorescence analysis of U-2 OS cells was conducted to detect the changes in the levels of p62 and LC3-II in response to a 24 hr treatment with SFN (20 μM) in the presence of Nrf2 (siCtrl) or absence of Nrf2 (siNrf2).