Fig. 2: DDX3 inhibition induces IRF3 phosphorylation, IFNβ expression and cell death in J-Lat 11.1 cells. | Nature Communications

Fig. 2: DDX3 inhibition induces IRF3 phosphorylation, IFNβ expression and cell death in J-Lat 11.1 cells.

From: Selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir

Fig. 2

a J-Lat 11.1 cells were transduced with a control lentiviral vector expressing a scrambled shRNA (shControl) or with lentivirus expressing shRNA against DDX3 (shDDX3) and relative DDX3 mRNA expression was quantified by RT-qPCR. Error bars represent the mean ± SD of three independent experiments (unpaired, two-tailed t-test; p value = 0.001038). b Representative Western blots depicting DDX3 and GAPDH in shControl and shDDX3 conditions. c The % of reactivation monitored as GFP production from cells as treated in a were quantified by Flow cytometry. Gating strategy as depicted in Supplementary Fig. 1a. Error bars represent mean ± SD of three independent experiments with at least 10,000 cells counted per treatment (paired, two-tailed t-test). d In cells treated as in a, relative GFP, vRNA, IL-10, IL-8, MMP9, IRF3 and IFNβ mRNA expression levels in shDDX3 treated cells were quantified by RT-qPCR and normalised to the shControl. Error bars represent mean ± SD of three independent experiments (unpaired, two-tailed t-test). e RK-33-treated J-Lat cells were sorted into GFP− (vRNA low) and GFP+ (vRNA high) fractions and the relative mRNA expression of vRNA using primers against (f) the vRNA and (g) RIG-I and MAVS expression were quantified by RT-qPCR. h Representative Western blots depicting phosphorylated IRF3, total IRF3 and GAPDH protein expression in RK-33-treated vRNA low and vRNA high populations. i Quantification of the densitometric analysis of P-IRF3 to GAPDH protein expression ratios from 2 h. Error bars represent mean ± SD from three independent experiments (unpaired, two-tailed t-test). The relative expression of (j) IFNβ mRNA and (k) ratios of Bcl2 and Bax mRNA expression in RK-33-treated vRNA low and vRNA high populations were quantified by RT-qPCR. Error bars represent mean ± SD from three independent experiments (unpaired, two-tailed t-test). l % Cell death was measured in Jurkat vs. J-Lat cells treated with increasing concentrations of RK-33 by flow cytometry. Error bars represent the mean ± SD of at least three independent experiments with at least 10,000 cells counted per treatment (paired, two-tailed t-test).

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