Fig. 4.
IKK2 deficiency differentially affects gene expression of naive and arthritic SFs. a Representative immunodetection of IKK2 levels in TAT-Cre treated Ikk2f/f (Ikk2Δ/Δ) and TAT-Cre treated hTNFtg Ikk2f/f (hTNFtg Ikk2Δ/Δ) SFs compared to non-treated control cultures Ikk2f/f and hTNFtg Ikk2f/f, respectively (upper panel). b Representative MTT incorporation assay in the indicated genotypes; hTNF quantitation in the supernatants of hTNFtg Ikk2Δ/Δ (grey column) and control hTNFtg Ikk2f/f SF cultures (black column) (n = 7, 2 experiments); flow cytometric detection of Vcam-1 and Icam-1 levels in cultured hTNFtg Ikk2f/f (black), hTNFtg Ikk2Δ/Δ (grey) and control SFs (Ikk2f/f: dotted black and Ikk2Δ/Δ: dotted grey) (n = 4, 2 experiments). c Representative immunodetection of Casp8 (C8), Cleaved Casp3 (CC3), Parp1, Sod2, Xiap, cIAP-1 and cFLIP(L) in cell extracts of Ikk2Δ/Δ, hTNFtg Ikk2Δ/Δ and control SF cultures treated with TNF (20 ng/ml, 5 h) (n = 10). d Relative gene expression of the NFκB-regulated genes Il6, Mmp-3, -13, -14, Timp1, Il1b and Tnfsf11 (RankL) (normalized to b2m levels) in SFs of the indicated genotypes upon TNF (20 ng/ml, 5 h) (n = 3–8). e Survival rates of Ikk2Δ/Δ and hTNFtg Ikk2Δ/Δ SFs treated with TNF, zVAD, Nec1s and GW806742X (GW) as indicated (n = 3). f Representative immunodetection of pMlkl, Mlkl, Ripk3, cFLIP(S) and IKK2 levels in TNF-treated Ikk2Δ/Δ, hTNFtg Ikk2Δ/Δ and control SF cultures (TNF: 20 ng/ml, 5 h) (n = 8, 4 experiments). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, by two-tailed Student’s t-test (b, d) or one-way ANOVA (e). Unprocessed original scans of blots are shown in Supplementary Fig. 8