Fig. 6: NLRX1-mediated ROS controls NF-κB signalling and affects cullin 1 NEDDylation.

Immunofluorescence microscopy of A549 cells stained with antibody for the p65 NF-κB subunit. Cells were pre-treated with mitoTEMPO (10 μM, 2 h pre-infection), or with vehicle control (DMSO) and infected with Kp52145 for 3 h (a). b Cells were transfected with NLRX1 siRNA (50 nM) or a non-silencing control (AS) and infected with Kp52145 for 3 h. c Cells were infected with Kp52145, the vgrG4 mutant (ΔvgrG4) or the complemented strain (comp, ΔvgrG4/pBAD30vgrG4) for 3 h. The percentage of p65 NF-κB localised in the nucleus is represented on the graph in a–c and it is the result of counting of minimum of hundred cells from each of three independent experiments. The total number of counted cells is indicated on top of each bar. ELISA of IL-8 secreted by A549 cells transfected with either NLRX1 siRNA (50 nM) or a non-silencing control (AS) and infected with K. pneumoniae strains. After 3 h of contact, the medium was replaced with medium containing gentamicin (100 µg/mL) to kill extracellular bacteria, and after 2 h the medium was collected (d). Images are representative of three independent experiments. Data in graphs are presented as the mean ± SD of three independent experiments. Two way-ANOVA with Holm-Sidak’s multiple comparisons test was used for statistical significance (p values indicated in the graph). e Immunoblot analysis of total IκBα and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. f Immunoblot analysis of phosphorylated IκBα and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. g Levels of phosphorylated Iκκα/β and tubulin in cells infected with Kp52145 for the indicated times. IκBα immunoprecipitation and immunoblot for K48-linkage specific polyubiquitin (Ub48) in cells treated with the proteasome inhibitor MG262 (5 μM, 2 h before infection), and infected with Kp52145 for 3 h or left uninfected (n.i.). Pre-immune mouse IgG was used as a control for immunoprecipitation (h). Immunoblot analysis of Cul-1 and tubulin levels in lysates of A549 cells infected with Kp52145 for 5 h, or treated with H₂O₂ (5 μM, 5 min). Cul-1 appears as a doublet, with the higher molecular band representing the NEDDylated form of Cul-1 (i). j Immunoblot analysis of Cul-1 and tubulin levels in lysates of A549 cells transfected with NLRX1 siRNA (50 nM) or a non-silencing control (AS), infected with Kp52145 for 5 h. k Cells were infected with Kp52145, the vgrG4 mutant (ΔvgrG4) or the complemented strain (comp) for 5 h, and the levels of Cul-1 and tubulin assessed by western blot. l Cells were pre-treated with mitoTEMPO (10 μM, 2 h pre-infection) or a vehicle control (DMSO), infected with Kp52145 for 5 h, and Ubc12 was immunoprecipitated followed by detection of NEDD8 and Ubc12 levels by immunoblotting. Images are representative of three independent experiments.