Figure 5

CD44 was essential for the MSC- and TSG-6-mediated anti-inflammatory responses. (A, B) BV2 cells, CD44-siRNA-BV2 cells, and control-siRNA-BV2 cells were incubated for 6 hours with LPS and with or without activated MSCs or TSG-6. The mRNA levels of TNF-α and IL-1β were measured by qRT-PCR. All transcriptional levels were normalized to GAPDH mRNA levels and determined by the 2^-ΔΔCt method. Values are mean ± SD. n = 3; **P <0.01; significantly different from control BV2 cells or control-siRNA-BV2 cells. (C) NF-κB DNA-binding activities were measured by EMSA assay. BV2 cells, CD44-siRNA-BV2 cells, and control-siRNA-BV2 cells were incubated for 1 hour with LPS and with or without activated MSCs or TSG-6. The data are representative of at least three independent experiments. (D) Luciferase reporter assays for NF-κB transcriptional activity in BV2 cells, CD44-siRNA-BV2 cells, and control-siRNA-BV2 cells (conditions as in Figure 2D) Values are mean ± SD. n = 3; *P <0.05; significantly different from control BV2 cells or control-siRNA-BV2 cells. (E) Cell extracts were prepared from BV2 cells, CD44-siRNA-BV2 cells, and control-siRNA-BV2 cells incubated for 30 minutes with LPS and with or without activated MSCs or TSG-6, then subjected to immunoblot analysis using antibodies against the phospho- or total-forms of the three MAPKs. (F-H) Quantification of western blot data. The phospho:total ratios of p38 (F), JNK (G), and Erk (H) were determined by measurements of band intensities of each protein kinase and quantified as fold changes over the control (unstimulated BV2 cells). Values are mean ± SD. n = 3; **P <0.01; significantly different from LPS-treated cells. Abbreviations: CD44-siRNA-BV2, BV2 cells transfected with CD44 siRNA; control-siRNA-BV2, BV2 cells transfected with control siRNA; LPS, lipopolysaccharide; NF-κB, nuclear factor (NF)-Κb; EMSA, electrophoretic mobility shift assay.