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Fig. S1. NF-κB readout using A549 p65–GFP reporter cells automated image analysis.

A. For screening purposes a modified A549 cell line with a p65–GFP reporter construct (SIB01 cell line) was used to monitor the translocation of GFP into the nucleus upon activation with IL-1β. The ratio of GFP signal in the nucleus versus the cytoplasm was taken as the level of cell activation.

B. To quantify cell populations, first cells were fixed and stained with Hoechst 33342 and images acquired. Second the images were subjected to automated image analysis. Based on the Hoechst signal, cell nuclei were detected as main object ‘nucleus’. Based on the features area and signal intensity only for main objects that met nuclei-like criteria the subobjects ‘core’ and ‘ring’ were applied. Based on the ratio of the measured GFP level for these subobjects, each main object was classified as non-activated (ratio < 0.9), activated (ratio > 1.1) or intermediate.

C. Example images for IL-1β-treated cells, and IL-1β and Kp52145-treated cells.

Fig. S2. Knockdown of some validated targets does not affect K. pneumoniae-induced ERK phosphorylation. Immunoblot analysis of P-ERK and tubulin levels in lysates of siRNA-transfected A549 cell infected with Kp52145 for the indicated times. siRNA targets are indicated (top row of each panel; AS-, AllStars control siRNA). Data are representative of at least three independent experiments.

Fig. S3. Effect of AKT inhibitor on Klebsiella pneumoniae-induced phosphorylations of ERK and GSK3β. Immunoblot analysis of P-ERK, P-GSK3β and tubulin levels in lysates of AKT inhibitor (AKT X, 30 μM, added 1 h before infection) or vehicle solution (DMSO)-treated A549 cells infected with Kp52145 for the indicated times. In all panels data are representative of at least three independent experiments.

Fig. S4. Effect of TLR2 and TLR4 on Klebsiella pneumoniae-triggered degradation of IκBα in CYLD knockdown cells. Immunoblot analysis of IκBα levels in lysates of siRNA-transfected (AS-, AllStars control siRNA; TLR2 siRNA, TLR4 siRNA, CYLD siRNA, CYLD and TLR2 siRNAs; and CYLD and TLR4 siRNAs) A549 cells which were infected with Kp52145 for 3 h or left uninfected. Membranes were reprobed for tubulin as a loading control. In all panels data are representative of at least three independent experiments.

Fig. S5. Effect of SRC inhibitor on Klebsiella pneumoniae-induced phosphorylation of EGFR. Immunoblot analysis of P-EGFR and total EGFR levels in lysates of SRC inhibitor (PP2, 40 μM, added 1 h before infection) or vehicle solution (DMSO)-treated A549 cells infected with Kp52145 for 3 h (Kp), challenged with purified CPS (100 μg ml−1) for 3 h (CPS) or left untreated (−). Data are representative of three independent experiments.

Fig. S6. Effect of CPS purified from K. pneumoniae 52145 on NF-κB activation. Immunofluorescence microscopy of A549 cells grown on glass coverslips. Cells were treated with CPS (100 μg ml−1) for 3 h (+ CPS) or left untreated (− CPS) (mock), and then stimulated with IL-1β (10 ng ml−1) for 20 min. Coverslips were stained with anti-p65 NF-κB subunit and rhodamine Red-X-conjugated donkey anti-rabbit antibody. Scale bar, 20 μm.

Fig. S7. Purified CPS from K. pneumoniae NTUH-K2044 activates the EGFR–PI3K–AKT–ERK–GSK3β signalling cascade.

A. Immunoblot analysis of P-EGFR and total EGFR levels in lysates of A549 cells infected with NTHU-K2044 (NTUH; wild-type strain) or treated for 3 h with 100 μg ml−1 CPS purified from NTHU-K2044.

B. Immunoblot analysis of P-EGFR and total EGFR levels in lysates of siRNA-transfected (AS-, AllStars control siRNA; TLR4 siRNA) A549 cells infected with NTHU-K2044 (NTUH) for 3 h or challenged with purified CPS (100 μg ml−1) for 3 h.

C. Immunoblot analysis of P-EGFR and total EGFR levels in lysates of siRNA-transfected (AS, AllStars control siRNA; c-SRC siRNA) A549 cells infected with NTUH-K2044 (NTUH) for 3 h or challenged with purified CPS (100 μg ml−1) for 3 h.

D. Immunoblot analysis of P-Akt, P-ERK, P-GSK3β levels and tubulin levels in lysates of A549 cells treated with EGFR inhibitor (AG1478, 5 μM, added 2 h before infection) or vehicle solution (−; DMSO); and infected with NTHU-K2044 (NTUH) for 3 h or challenged with purified CPS (100 μg ml−1) for 3 h.

E. Immunoblot analysis of CYLD and tubulin levels in lysates of A549 cells infected with NTHU-K2044 (NTUH) for 3 h or challenged with purified CPS (100 μg ml−1) for 3 h.

F. Immunofluorescence microscopy of A549 cells grown on glass coverslips. Cells were treated with CPS (100 μg ml−1) for 3 h (CPS); infected with NTHU-K2044 (NTHU) or left untreated (−) (mock), and then stimulated with IL-1β (10 ng ml−1) for 20 min. Coverslips were stained with anti-p65 NF-κB subunit and rhodamine Red-X-conjugated donkey anti-rabbit antibody. Scale bar, 20 μm.

Fig. S8. siRNA knockdown efficiency.

A. Immunoblot analysis of EGFR, MyD88, ERK, CYLD, PAK4, c-SRC levels in lysates of siRNA-transfected (AS, AllStars control siRNA; EGFR siRNA, MyD88 siRNA, ERK siRNA, CYLD siRNA, PAK4 and c-SRC siRNA) A549 cells. Membranes were reprobed for tubulin as a loading control.

B. Transcript levels of indicated targets relative to AllStars control siRNA-transfected cells, as determined by RT-qPCR and normalization to GAPDH transcript levels.

C. Levels of ERK1 and ERK2 transcripts in ERK knockdown cells relative to AllStars control siRNA-transfected cells, as determined by RT-qPCR and normalization to GAPDH transcript levels.

Data are representative of three independent experiments in all panels.

Table S1. Targets selected for hit validation.

Table S2. Description of reagents (siRNA, primers and antibodies) used in this study.

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