Sb202190

PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 (InvivoGen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 (InvivoGen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, InvivoGen), SB202190 (p38α/β MAPK inhibitor, InvivoGen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in Supplementary Figure 2. For viability analysis, cells were treated as indicated, stained for dead cells using the fixable viability dye eFlour780 (#65-0865-14, eBioscience), and measured using a BD LSRFortessa™ flow cytometer (BD Biosciences). Data were analyzed using FlowJo V.7 (Treestar Inc., Ashland, OR, USA) and GraphPad PRISM (GraphPad Software, San Diego, California, USA).

HEK293 Flp pFRT/eGFP was a gift from Jacob Giehm Mikkelsen, Aarhus University, while CRC cells originated from the ATCC and NCI-60 repositories (kind gift from Nils Brünner, University of Copenhagen). The cell lines were authenticated by our in-house STR analysis (http://identicell.dk), and were tested negative for mycoplasma using MycoSensor PCR Assay Kit (Stratagene). All the cell lines were grown in RPMI medium 1640 with L-glutamine (Life Technologies) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies). The cells were propagated in 37 °C at 90% air humidity and with 5% CO₂. For oxPt treatment cells were first induced for 48 or 72 h with 50 ng ml⁻¹ doxycycline hyclate (Sigma-Aldrich), and then cultured in medium supplemented with the indicated concentrations of oxPt (Fresenius Kabi) together with doxycycline. Chemical inhibitors SB203580 (Invivogen) and SB202190 (Invivogen) were dissolved in dimethyl sulphoxide (DMSO) and kept in aliquots at −20 °C until use. The cells were pre-incubated 1 h with inhibitor (or DMSO) supplemented medium before exposure to oxPt (or medium) containing inhibitor (or DMSO).