Fig. 6: TNF protects RA fibroblasts from the insult of lipid peroxidation and ferroptosis by enhancing the GSH biosynthetic pathway via activation of NF-κB.

a Intracellular GSH in fibroblasts treated with TNF in the presence of adalimumab followed by IKE treatment for 18 h. n = 3 biologically independent samples per condition. *P = 0.0211, 0.0224, 0.0109 (left to right); two-tailed t-test. b Western blotting analysis of GCLC, GCLM, and SLC7A11 expression in fibroblasts at 24 h and 72 h after exposure to TNF and adalimumab. c Western blotting analysis of SLC7A11 in fibroblasts transfected with scramble siRNA or either of two independent siRNAs targeting SLC7A11 (siSLC7A11#1, siSLC7A11#2). d Relative cell viability and cell death of fibroblasts transfected with scramble siRNA or siRNA targeting SLC7A11, primed with TNF and then treated with RSL3. n = 3 biologically independent samples per condition. ****P < 0.0001, ***P = 0.0003, *P = 0.0101; one-way ANOVA followed by multiple comparisons. e Relative viability of fibroblasts from individuals with RA and primed with TNF in the presence of the GCLC inhibitor BSO, followed by treatment with IKE (1 μM) or RSL3 (0.125 μM). ***P = 0.0001, *P = 0.0167; two-tailed t test. f Western blotting analysis of GCLC expression in fibroblasts transfected with scramble siRNA or either of two independent siRNAs targeting GCLC (siGCLC#1, siGCLC#2). g Relative viability and cell death of fibroblasts transfected with scramble siRNA or siRNA targeting GCLC, primed with TNF, and then treated with RSL3. Cell viability was assayed at 12 h, and cell death was measured at 10 h. n = 3 biologically independent samples per condition. Left, ns, P = 0.2174, *P = 0.0314; right, ns, P = 0.7042, **P = 0.0064; one-way ANOVA followed by multiple comparisons. h Western blotting analysis of NF-κB p65 expression in fibroblasts transfected with scramble siRNA or either of two independent siRNAs targeting NF-κB p65 (siNF-κB#1, siNF-κB#2). i Relative viability and cell death of fibroblasts transfected with scramble siRNA or siRNA targeting NF-κB p65, primed with TNF and then treated with RSL3. n = 3 biologically independent samples per condition. Left, ns, P = 0.7675, **P = 0.0079; right, ns, P = 0.9997, **P = 0.0099; one-way ANOVA followed by multiple comparisons. j Western blotting analysis of NF-κB p65, GCLC, GCLM, and SLC7A11 expression in fibroblasts transfected with scramble siRNA or siRNA targeting NF-κB p65 and treated with TNF for 72 h. k Western blotting analysis of NF-κB p65, p-IκB, IκB, GCLC, and GCLM expression in fibroblasts treated with TNF and the IκB kinase inhibitor PS1145 for 72 h. l Relative NF-κB activity in fibroblasts treated with TNF and PS1145. n = 3 biologically independent samples per condition. *P = 0.0263, 0.0123 (left to right); one-way ANOVA followed by multiple comparisons. m Relative viability and cell death in fibroblasts primed with TNF and PS1145 and then treated with RSL3 and ferrostatin-1. n = 3 biologically independent samples per condition. ****P < 0.0001; one-way ANOVA followed by multiple comparisons. n Lipid ROS levels in fibroblasts primed with TNF and PS1145 and then treated with RSL3 and ferrostatin-1. n = 3 biologically independent samples per condition. ****P < 0.0001; one-way ANOVA followed by multiple comparisons. o Intracellular GSH in fibroblasts treated with TNF in the presence of PS1145. n = 3 biologically independent samples per condition. ***P = 0.0001, ****P < 0.0001; one-way ANOVA followed by multiple comparisons. p ROS levels in fibroblasts primed with TNF and NOX inhibitor (NOXi) for 6 or 48 h. n = 2 biologically independent samples per condition. *P = 0.0469; two-tailed t test. q Relative cell death in fibroblasts primed with TNF and NOXi for 48 h and then treated with RSL3. n = 3 biologically independent samples per condition. **P = 0.0083, ***P = 0.0003; two-tailed t test. All bar graphs are presented as mean ± SD. Source data are provided as a Source data file.