Figure 4.
NF-κB activation is required for mesoangioblast migration toward HMGB1 and CXCL12, but not other chemoattractants. (A) 100 ng/ml HMGB1 promotes nuclear translocation of p65 in mesoangioblasts. Paired images show DAPI (blue) and p65/RelA (detected via indirect immunofluorescence; red). Stimulation with 20 ng/ml TNF-α is shown as positive control. (B) 100 ng/ml HMGB1 and 20 ng/ml TNF-α induce NF-κB–dependent transcription of the endogenous IκBα gene in mesoangioblasts. Absolute quantities are normalized to β-actin. Results are shown as the mean of duplicate samples ± SD and were replicated three times. (C) 100 ng/ml SDF-1/CXCL12 promote nuclear translocation of p65 in mesoangioblasts (shown as in A). (D) Mesoangioblast migration toward HMGB1 depends on NF-κB activation. Mesoangioblasts were transfected with vectors encoding IκBαSR and GFP (pMP9IκBαSR) or GFP alone (pMP9), grown for 48 h, and induced to migrate for 6 h in Boyden chambers toward 1% serum (FCS), 100 ng/ml HMGB1, 10 ng/ml PDGFBB, 0.1 nM fMLP, or no chemoattractant (control). Only fluorescent cells were counted. Results are the mean of triplicate samples ± SD, and were replicated three times. *, P < 0.05. (E) Mesoangioblast migration toward 100 ng/ml CXCL12, but not 20 ng/ml TNF-α, depends on NF-κB activation. Results are the mean of triplicate samples ± SD; experiments were performed as described in D.