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. 2022 Feb 3;13:676. doi: 10.1038/s41467-021-27948-4

Fig. 7. A TNF antagonist sensitizes RA fibroblasts to ferroptosis induction in CIA model mice.

Fig. 7

a Three-dimensional spheroids formed by fibroblasts were primed with TNF and the TNF antagonist etanercept for 48 h before treatment with IKE (4 μM) for an additional 30 h. Dead cells were stained with SYTOX and Hoechst33342 for 1 h. Scale bars, 100 μm. b Relative viability of fibroblasts from patients with RA that were primed with fibroblast supernatant in the presence of the TNF antagonist etanercept for 48 h followed by treatment with IKE (1 μM) and ferrostatin-1 (Fer1, 1 μM) for an additional 26 h. n = 3 biologically independent samples per condition. **P = 0.0092; two-tailed t test. c, d CIA model mice were intraperitoneally injected with 20 mg/kg IKE twice a week and/or 2 mg/kg etanercept twice a week for 5 weeks. Joint inflammation measured by arthritis score (c) (*P = 0.0461, CIA + Etanercept vs CIA + Etanercept+IKE; **P = 0.0132, CIA + IKE vs CIA + Etanercept+IKE; one-way ANOVA followed by multiple comparisons to compare the means at the end point) and paw thickness (d) (*P = 0.0233, CIA + Etanercept vs CIA + Etanercept+IKE; *P = 0.0356, CIA + IKE vs CIA + Etanercept+IKE; one-way ANOVA followed by multiple comparisons to compare the means at the end point). n = 5 mice for each group. e H&E staining images of representative joints in control and day 36 CIA model mice. Scale bars, 200 μm. f Representative micro-CT images of control and CIA mice treated with IKE and/or etanercept. g Toluidine blue O and safranin O staining images of representative joints. Scale bars, 200 μm. h Quantification of histomorphometric analysis of cartilage damage, bone erosion, and pannus formation. Left, *P = 0.0447, **P = 0.0011, ****P < 0.0001; middle, *P = 0.0150, **P = 0.0014; right, *P = 0.0152, **P = 0.0099, ****P < 0.0001; one-way ANOVA followed by multiple comparisons. n = 10 joints for each group. i Representative fluorescent multiplex IHC staining and quantification of joints labeled with anti-F4/80 (red), anti-FAPα (green), and DAPI (blue). Scale bars, 100 μm. Etan, Etanercept. j Immunohistochemical staining of p-NF-κB, GCLM, and GCLC in the joints of CIA model mice with or without etanercept treatment. Scale bars, 25 μm. k Immunohistochemical staining and scoring of PTGS2 and GPX4 expression in the joints of CIA mice. n = 15 joints for each group. *P = 0.0425, ****P < 0.0001; one-way ANOVA followed by multiple comparisons. Scale bars, 50 μm. Data in c and d are presented as mean ± SEM. Other data are presented as mean ± SD. Source data are provided as a Source data file.