For inhibition experiments mDCs were pre-incubated with the indicated amounts of rapamycin (mTOR inhibitor), BAY-11-7082 (irreversible inhibitor of TNF-α-induced IkB-α phosphorylation resulting in inactivation of NFkB), triptolide (NFκB inhibitor), dexamethason (NFκB and MAPK inhibitor), the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) and BMS-345541 (Abcam, Cambridge, UK), as well as the MAPK inhibitors SP600125 (JNK MAPK inhibitor, Invivogen, Toulouse, France), SB-202190 (p38α/β MAPK inhibitor, Invivogen, Toulouse, France), or U0126 (MEK1/2 MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with equimolar amounts of rFlaA + rBet v 1 or rFlaA:Betv1 for either 30 min (Western Blot), 24 h (ELISA and cytotoxicity), or 72 h (ELISA and analysis of cell metabolic state). Toxicity of the used inhibitors was determined using the fixable viability dye eFlour780 (Thermo Fisher Scientific, Darmstadt, Germany, Repository Figure S1 ). Inhibitor concentrations showing toxic effects were excluded from the analysis.
Bms 345541
Manufactured by Abcam
Sourced in United Kingdom
BMS-345541 is a selective IKK-β inhibitor. It inhibits the phosphorylation and degradation of IκBα, thereby blocking the activation of NF-κB.
Automatically generated - may contain errors
Lab products found in correlation
Eflour780, by Thermo Fisher Scientific (2 mentions)
Sb202190, by InvivoGen (2 mentions)
Tpca 1, by Abcam (2 mentions)
Sp600125, by InvivoGen (2 mentions)
U0126, by Cell Signaling Technology (2 mentions)
Sb202190, by Merck Group (2 mentions)
Psuper retro neo gfp vector, by Oligoengine (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Sp600125, by Merck Group (2 mentions)
Lenticrispr v2, by Addgene (2 mentions)
Lipofectamine 2000 and 3000, by Thermo Fisher Scientific (2 mentions)
Bay 11 7082, by Abcam (1 mentions)
12 well non treated plate, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Facsuite software, by BD (1 mentions)
Fitc brdu flow kit, by BD (1 mentions)
Akt inhibitor x, by Cayman Chemical (1 mentions)
Ct rmc4550, by Chemietek (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
8 protocols using bms 345541
1
Inhibiting Immune Response Pathways
2
Inhibition of NF-κB Signaling in BMDCs
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The NF-κB signaling pathway was inhibited by 1 h pre-treatment with the IKKβ inhibitor IMD-0354 (IC50 = 250 nM, ab144823, Abcam) at three different concentrations (100, 250, and 500 nM) and with the IKKβ and IKKα inhibitor BMS-345541 (IC50 of 0.3 and 4 µM, respectively, ab144822, Abcam) at 5 µM. Stock solutions of the inhibitors at 25 mM were prepared in DMSO following manufacturer’s recommendations, further diluted in PBS pH 7.2 and added to complete BMDC medium as PBS-diluted solutions to achieve the concentrations analyzed. The vehicle control for the inhibitors constituted the least diluted DMSO:PBS solution (1:2). As a negative control, both vehicle controls (for lipids and inhibitors) were combined and added to the cells at equal volumes as their tested counterparts. The inhibitors alone were also assessed throughout the duration of the assay. L. johnsonii N6.2 lipids were added as TLs or PLs at 0.5 or 5 µg/mL and cells were stimulated for 6 additional hours. 3∙105 7-d BMDCs per well were assayed in 1 mL of complete BMDC medium in a 12-well non-treated plate (Thermo Scientific).
3
Knocking Down TAK1 in NMuMG Cells
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For knocking down TAK1 in NMuMG cells, pSuper retro-neo-GFP vector (OligoEngine, Seattle, WA) was used to construct shTAK1 plasmid that targets 3’-UTR of mouse TAK1 gene. Flag-tagged mouse TAK1FL or TAK1∆E12 plasmids were constructed by insert TAK1 cDNA from MGC clone 5989 (Image: 3499247) or HA-TAK1 (gift from Dr. K. Matsumoto) (10 (link)) to pLNCX vector, respectively. For CRISPR/Cas9 mediated editing of the TAK1 gene, plasmid lentiCRISPRv2 (Addgene 52961) was used to deliver individual sgRNAs (single guide RNA). Sequences of primers used in the construction of shRNA, cDNA and CRISPR/Cas9 vectors are listed in Supplementary Table S1 . Stocks of lentiviruses were made after co-transfecting lentiCRISPRv2, pVSVG and psPAX2 plasmids into HEK293T cells. Lipofectamine 2000 and 3000 (Life Technologies, Grand island, NY) was used to transfect plasmid DNA according to the manufacturer’s instructions. G418 or puromycin (Life Technologies) was used to select stable clones. JNK inhibitor, SP600125, and p38 inhibitor, SB202190 were obtained from EMD Millipore (Burlington, MA), NF-kB inhibitor, BMS345541, was obtained from Abcam (Cambridge, MA)
4
Osteoclast Formation and Function Assays
5
Sphere Formation Assay for BTICs
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For the sphere formation assay, freshly dissociated cells were plated at 10,000 cells per well in 100 μl of BTIC medium into a 96-well flat bottom plate. The resultant number of spheres above the 60-μm-diameter cutoff, a convenient parameter to describe growth characteristics, was monitored after 3 to 5 days by bright-filed photographing multiple fields per well with subsequent analyzes as previously described (8 (link), 43 (link), 58 (link)). For the blockade of signaling pathways, BTICs were treated with the following inhibitors: p38 MAP Kinase Inhibitor Skepinone-l (Millipore, 506174), IKK-α/β inhibitor BMS 345541 (Abcam, ab144822), or RMC-4550 (Chemietek, CT-RMC4550).
6
Inhibition of Inflammatory Pathways in THP-1 Macrophages
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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
7
Evaluating Epithelial-Mesenchymal Transition in Cells
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The reagents used were as follows: OVA (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), acetylcholine chloride (Ach; Sigma-Aldrich; Merck KGaA), phosphate-buffered saline (PBS; Sigma-Aldrich; Merck KGaA), sodium pentobarbital (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), BMS-345541 (Abcam, Cambridge, MA, USA), E-cadherin antibody (cat. no. ab76055; Abcam), vimentin antibody (cat. no. ab92547, Abcam), β-actin antibody (cat. no. ab179467; Abcam), glyceraldehyde 3-phosphate dehydrogenase (GADPH; Sigma-Aldrich; Merck KGaA), dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA), diaminobenzidine (DAB; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), an enzyme-linked immunosorbent assay (ELISA) kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), Pierce Enhanced Chemiluminescence (ECL) western blot substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA), a reverse transcription kit (Promega Corporation, Madison, WI, USA), a quantitative polymerase chain reaction (qPCR) kit (TransStart Green; Beijing Transgen Biotech Co., Ltd., Beijing, China), a bicinchoninic acid (BCA) protein assay kit and radioimmunoprecipitation assay (RIPA) lysis buffer (Beijing ComWin Biotech Co., Ltd., Beijing, China). The qPCR primers were designed and synthesized by Nanjing Kingsy Biotechnology (Nanjing, China).
8
Knocking Down TAK1 in NMuMG Cells
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