Fig. 5: Inhibition of the STAT1 and NF-κB signaling pathways is responsible for the CMA-mediated inhibition of MSC immunosuppressive function.

a SCR-MSCs and L2A-KD-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL each) for the indicated time. Cells were harvested, and NF-κB p65, STAT1, and phosphorylation of NF-κB p65 and STAT1 at Tyr701 were analyzed by immunoblotting. The relative densitometry of p-NF-κB p65 and p-STAT1 (Tyr701) was quantified using ImageJ software and was compared to β-actin. b SCR-MSCs and L2A-KD-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL each) for 6 h. Nuclear proteins were harvested. NF-κB p65 and STAT1 were analyzed by immunoblotting. c–g After pretreatment with DMSO, fludarabine (2 μM), or BAY11-7082 (10 μM) for 6 h, SCR-MSCs and L2A-KD-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL each) for 24 h. Protein, mRNA, and supernatant were collected. The expression of iNOS was measured at the protein, and mRNA levels using immunoblotting and quantitative real-time PCR, respectively c–e. The concentration of NO in the culture supernatant was detected by Griess reagent f. CXCL10 mRNA expression was detected by quantitative real-time PCR g. h, i SCR-MSCs and L2A-KD-MSCs were pretreated with DMSO, fludarabine (2 μM), or BAY11-7082 (10 μM) for 6 h and then treated with mitomycin C (50 ng/mL) for 4 h prior to coculture with CFSE-labeled splenocytes activated by anti-CD3/CD28 antibodies for 3 days at a ratio of 1:20. CD8⁺ and CD4⁺ T cells were stained for proliferation analysis by flow cytometry at the end of coculture, and the percentage of nonproliferating T cells is shown. The results are representative of three to six independent experiments and are presented as the mean ± s.e.m. Significant differences were analyzed by one-way ANOVA h, i or two-way ANOVA a–g, and are expressed as *P < 0.05, ***P < 0.001, and n.s., no significance.