1. Date of download: 5/30/2016 Copyright © 2016 American Society of Anesthesiologists. All rights reserved. Fig. 4. Effects of human placental multipotent mesenchymal stromal cells (hPMSCs) on regulating nuclear factor-κB (NF-κB) activation in RAW264.7 cells. (A ) Representative gel photography and the densitometric analysis data of phosphorylated NF-κB p65 (p-NF-κB p65) protein concentrations in the nuclear extracts of RAW264.7 cells using immunoblotting assay. The p-NF-κB p65 protein concentrations were normalized by Histone-H3. (B ) Representative gel photography and the densitometric analysis data of cytosolic inhibitor of NF-κB (I-κBα) and phosphorylated I-κBα (p-I-κBα) protein concentrations in the cytosolic extracts of RAW264.7 cells using immunoblotting assay. The I-κBα and p-I-κBα protein concentrations were normalized by β-Actin. (C ) Representative findings of immunofluorescent staining assay for p-NF-κB p65 in RAW264.7 cells (i.e., NF-κB nuclear translocation). The p-NF-κB p65 protein was stained with fluorescent rhodamine isothiocyanate- conjugated antibody. Nuclei were counterstained with diamidino-2-phenylindole. The cells were imaged using a confocal microscope. (D ) Representative gel photography and densitometric analysis data of the NF-κB-DNA binding activity in the nuclear extracts of RAW264.7 cells using chemiluminescence electrophoretic mobility shift assay. Human placental MSCs (cell number: hPMSCs vs. RAW264.7 cells = 1:10 2 ) were added to RAW264.7 cells at 4 h before lipopolysaccharide (LPS) administration. NS- 398 (1 μM) was added at 30 min before hPMSCs. PBS+R is the RAW264.7 cells plus phosphate buffered saline group; LPS+R is the RAW264.7 cells plus LPS (100 ng/ml) group; LPS+R+S is the RAW264.7 cells plus hPMSCs plus LPS group; LPS+R+S+N is the RAW264.7 cells plus hPMSCs plus NS-398 plus LPS group. All data were derived from three independent experiments performed in duplicates (n = 6) and expressed as mean ± standard deviations. Immunoblotting assay data were analyzed by repeated measures ANOVA followed by multiple comparisons with the Bonferroni correction for the significance level. For immunoblotting assay data, * indicated significant differences (P < 0.017) between the LPS+R+S and the LPS+R groups or the LPS+R+S+N and the LPS+R groups, and # indicated significant differences (P < 0.017) between the LPS+R+S+N and the LPS+R+S group. EMSA data were analyzed with one-way ANOVA with the Tukey test for post hoc comparisons. For EMSA data, * indicated significant differences (P < 0.05) between the LPS+R+S and the LPS+R groups, and # indicated significant differences (P < 0.05) between the LPS+R+S+N and the LPS+R+S group. I-κBα = inhibitor of NF-κB; LPS = lipopolysaccharide; N = NS-398; NF-κB = nuclear factor-κB; p-I-κBα = phosphorylated I-κBα; p-NF-κB p65 = phosphorylated NF-κB p65; PBS = phosphate buffered saline; R = RAW264.7; S = human placental multipotent mesenchymal stromal cells, or hPMSCs. Figure Legend: Antiinflammation Effect of Human Placental Multipotent Mesenchymal Stromal Cells Is Mediated by Prostaglandin E 2 via a Myeloid Differentiation Primary Response Gene 88-dependent Pathway Anesthesiology. 2012;117(3):568-579. doi:10.1097/ALN.0b013e31826150a9 Antiinflammation Effect of Human Placental Multipotent Mesenchymal Stromal Cells Is Mediated by Prostaglandin E 2 via a Myeloid Differentiation Primary Response Gene 88-dependent Pathway Anesthesiology. 2012;117(3):568-579. doi:10.1097/ALN.0b013e31826150a9