Fig. 3

3R4F-exposure induces ROS in hMSCs, resulting in a reduction in the supporting ability of HSPCs. (A) mRNA expression levels of ROS-related genes (AhR, CYP1A1, and NOS2) in 3R4F-treated hMSCs were analyzed by qRT‒PCR. The expression level of each gene was normalized to that of β-actin. (B-D) hMSCs were pretreated with or without NAC for 1 h and followed by treatment with 5% 3R4F for 72 h. (B) Intracellular ROS levels were measured by flow cytometric analysis by using the ROS-sensitive fluorophore 2’,7’-dichlorofluorescin diacetate (DCFDA). (C) The basal level of the cellular respiration rate (oxygen consumption rate [OCR]) was determined by using an XFe24 Extracellular Flux Analyzer. (D) mRNA expression level of HSPC niche related genes in 3R4F-treated MSCs were analyzed by qRT-PCR and normalized to that of β-actin. (E-G) hCD34⁺ HSPCs were cocultured with 3R4F-treated hMSCs with or without NAC pretreatment for 72 h. (E) After 3 days of coculture, the CD34⁺CD90⁺ HSPC population in CD45⁺ cells were analyzed using flow cytometry. (F, G) After coculture, HSPCs were seeded in methylcellulose colony formation medium and cultured for 2 weeks. (F) Representative colony morphologies (Scale bar: 200 μm). (G) The number of BFU-E, CFU-GM, CFU-GEMM, and the total sum of all colonies were quantified. (H) hMSCs were pretreated with or without NAC for 1 h and followed by treatment with 5% 3R4F for 48 h. mRNA expression levels of inflammation-related genes (IL-6, IL-8, TNF-α, cFOS, and IL-10) were determined by qRT-PCR. The expression level of each gene was normalized to that of β-actin. The data are presented as the mean ± S.D. of three independent experiments (*p < 0.05; **p < 0.01). BFU-E – Burst forming Erythrocyte; GM – Granulocyte/Macrophage; GEMM – Granulocyte/Erythrocyte/Macrophage/Megakaryocyte.