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. 2017 Oct 24;26(1):56–69. doi: 10.1016/j.ymthe.2017.10.014

Figure 3.

Figure 3

Vanadate Facilitates Virus-Induced Type I Interferon and ROS-Mediated Cell Death

(A) 786-0 cells were pre-treated with a range of concentrations of vanadate for 4 hr and were subsequently infected with VSVΔ51 expressing GFP at an MOI of 0.01. 786-0 cell viability was assayed in cells 24 hr post-infection. Results were normalized to the average of the values obtained for the corresponding uninfected, untreated cells (n = 4; error bars indicate SEM; significance decrease in viability at 16–1,000 μm; ***p < 0.0001 by two-way ANOVA; as compared to uninfected condition). (B) 786-0 were pre-treated with vanadate (100 μM) for 4 hr and subsequently infected with oncolytic VSVΔ51 expressing GFP at an MOI of 0.01. Twenty-four hours post-infection, induction of cell death was determined by annexin V and 7-aminoactinomycin D (7-AAD) staining. Numbers indicate the percentage in each quadrant. (C) Cell lysates of 786-0 treated with vanadate (100 μM) and VSVΔ51 expressing GFP were collected at indicated time points, RNA was collected, and expression of PUMA and Noxa genes was quantified by qPCR (n = 3; error bars indicate SEM; **p < 0.01, ***p < 0.0001 by two-way ANOVA; comparing VSVΔ51 condition to vanadate + VSVΔ51 condition). (D and E) 786-0 cells were pre-treated with vanadate for 4 hr and subsequently infected with (D) VSVΔ51, UV-inactivated VSVΔ51, VSVΔ51ΔG, or (E) treated with IFNa, IFNb, or Poly I:C. Cell viability was assayed 48 hr post-infection or treatment. Corresponding cell morphology is presented in (F). (G–I) 786-0 cells were co-treated with vanadate and N-acetyl-L-cysteine (NAC) for 4 hr and infected with (G–I) VSVΔ51 expressing GFP at an MOI of 0.01 or with (H) IFNa, IFNb, or Poly I:C, and cell viability was assayed 48 hr post-infection or treatment. In (I), viral titers were determined 24 hr post-infection from supernatants (n = 3; error bars indicate SEM; ***p < 0.001 by two-way ANOVA; as compared to mock-treated condition). (J and K) MRC5 (J) and GM38 (K) normal cells were pre-treated with vanadate for 4 hr and subsequently infected with VSVΔ51 or treated with IFNa, IFNb, or Poly I:C. For (D), (E), (G), (H), (J), and (K), cell viability results were normalized to the average of the values obtained for the corresponding uninfected, untreated cells (n = 4; error bars indicate SEM; NS, no statistical significance, ***p < 0.0001 by two-way ANOVA).