Fig. 3.

NAMPT is required for the immunomodulatory effects of MSCs on T cells and macrophages. A Splenocytes were stained with CFSE and cocultured with EtOH-pretreated MSCs or FK866-pretreated MSCs (2.5 × 10⁴ cells per well in 48-well plates) at a ratio of 1:20 (MSC:splenocyte) for 72 h in the presence of anti-CD3 (1 μg/ml). The reduction in the CFSE fluorescence intensity in splenocytes was detected by flow cytometry (n = 4). B Bone marrow-derived macrophages (BMDMs) were treated with different MSC supernatants (MSC-S) for 48 h. The expression of anti-inflammatory genes Arg-1, Chil3, Cd206 and Tgf-β in mature BMDMs was determined by qRT‒PCR (n = 3). C BMDMs were treated with different MSC-S for 48 h and subsequently treated with LPS (100 ng/mL) for 24 h. The expression of the proinflammatory genes Il-6, Il-12 and Tnf-α in inflammatory macrophages was determined by qRT‒PCR (n = 3). D Splenocytes were stained with CFSE and cocultured with Scrambled-shRNA or Nampt-shRNA MSCs (2.5 × 10⁴ cells per well in 48-well plates) at a ratio of 1: 20 (MSC:splenocyte) for 72 h in the presence of anti-CD3 (1 μg/ml). The reduction in the CFSE fluorescence intensity in splenocytes was detected by flow cytometry (n = 4). E BMDMs were treated with different MSC-S for 48 h. The expression of the anti-inflammatory genes Arg-1, Chil3, Cd206 and Tgf-β in mature BMDMs was determined by qRT‒PCR (n = 3). F BMDMs were treated with different MSC-S for 48 h and subsequently treated with LPS (100 ng/mL) for 24 h. The expression of the proinflammatory genes Il-6, Il-12 and Tnf-α in inflammatory macrophages was determined by qRT‒PCR (n = 3). Values are presented as the mean ± SEM. Statistical analysis was performed by one-way analysis of variance