Figure EV1. Analysis of effect of Pseudomonas on NF‐κB translocation into the nucleus.

1. Images of treated/infected A549 cells were taken by confocal microscopy and analysed with a specific plugin developed in our team. NF‐κB was labelled with an anti‐p65 antibody (red) and nucleus with DAPI (blue). Rows correspond to (i) raw images, (ii) after median filter, (iii) treated with threshold moments, (iv) after subtraction of total NF‐κB from DAPI to obtain the cytoplasmic NF‐κB and (v) after subtraction of total NF‐κB from NF‐κB cytoplasmic to obtain nuclear NF‐κB.
2. Control experiment in which A549 cells were either mock infected, treated with TNFα (1 μg/ml) or infected with either heat‐killed (HK) P. aeruginosa PA7, wt, ∆pumA, ∆pumA::pumA (Ara) induced with 1% arabinose. After 1 h cells were fixed, labelled as described in Materials and Methods, images taken by confocal microscopy and analysed with the plugin. Between 200 and 400 cells per condition were counted and data correspond to median ± standard error from three independent experiments. Non‐parametric, one‐way ANOVA, Kruskal–Wallis test was performed, with Dunn's multiple comparisons test, ****P < 0.0001, ***P < 0.001, *P < 0.05.