Fig. 5

3R4F-induced ROS impair hematopoiesis supportive ability of hMSCs via the NLRP3 inflammasome. (A) An illustration of 3R4F-induced ROS activation and the NLRP3 expression-related pathway was depicted based on the DEG profile of hMSCs. IPA software was used for in silico analysis. (B, C, D) hMSCs were pretreated with or without NAC or MCC950 for 1 h and followed by treatment with 5% 3R4F for 72 h. (B) The mRNA expression levels of NLRP3 inflammasome related genes (NLRP3, PYCARD, and IL-1β) was analyzed by qRT‒PCR. The expression level of each gene was normalized to that of β-actin. (C) mRNA expression level of inflammation related genes (NLRP3, CASP1, IL-1β, and IL-10) were analyzed by qRT‒PCR. The expression level of each gene was normalized to that of β-actin. (D) The expression of NLRP3, ASC, pro/cleaved Caspase-1, and pro/cleaved IL-1β was analyzed and quantified by western blotting. (E) MCC950-treated hMSCs were cocultured with hCD34⁺ HSPCs for 3days. CD34⁺CD90⁺ HSPC population in CD45⁺ cells was analyzed using flow cytometry. (F) Three days after coculture, HSPCs were seeded in methylcellulose colony formation medium and cultured for 2 weeks. The number of BFU-E, CFU-GM, CFU-GEMM, and the total sum of all colonies were quantified. Data are presented as the mean ± S.D. (*p < 0.05; **p < 0.01; ***p < 0.001). BFU-E – Burst forming Erythrocyte; GM – Granulocyte/Macrophage; GEMM – Granulocyte/Erythrocyte/Macrophage/Megakaryocyte.