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. 2019 Oct 24;10:2431. doi: 10.3389/fimmu.2019.02431

Figure 5.

Figure 5

Depletion of endogenous NLRX1 lead to increase of apoptosis and innate immune response. (A) PMVECs were transfected with 50 nM NLRX1, TBK1, or control siRNA for 72 h, and treated with vehicle or sodium azide for 2 h before cell lysis. Cell lysates were subjected to SDS-PAGE and immunoblotting using NLRX1, p-IKK, IKK, p-TBK1, TBK1, caspase 3, and PARP1 antibodies. GAPDH was used as the loading control. There were two samples each group, the blots shown are representative of the replicates (n = 4). (B) PMVECs were transfected with 50 nM NLRX1 or control siRNA, 72 h after the transfection; cells were fixed with iced methanol and stained for NLRX1 and DNA. Scale bar: 20 μm. The data shown is representative of the replicates (n = 5). (C) PMVECs were transfected with 50 nM NLRX1 or control siRNA for 72 h, and treated with sodium azide for indicated period of time before lysis. Cell lysates were subjected to SDS-PAGE and immunoblotting using NLRX1, p-IKK, IKK, p-TBK1, TBK1, p-P65, and P65 antibodies. GAPDH was used as the loading control. The blots shown are representative of the replicates (n = 3). (D) PMVECs were transfected with 50 nM NLRX1 or control siRNA for 72 h, and pre-stained with MitoTracker Red CMXRos for 30 min. Cells were fixed with iced methanol and stained for p-TBK1 and DNA. Scale bar: 10 μm. The data shown is representative of the replicates (n = 5). (E) SYBR green real-time PCR amplification of IL-6, IL-10, IL-1β, TNF-α, IL-18, and IFN-β. PMVECs were transfected with 50 nM NLRX1 or control siRNA for 72 h, and treated with vehicle or sodium azide for 3 h. * indicates p < 0.05 compared to control siRNA group, # indicates p < 0.05 compared to azide treated control siRNA group.