Fig. 5

MSC-H cells were activated by HMGB1 autocrine signaling. a Transfection of MSCs with HMGB1 overexpressing vectors and vehicle control vectors yielded MSC-H and MSC-C cells, respectively. Both of them expressed GFP, rendering them visible under a fluorescent microscope. The level of HMGB1 was 1.5-fold higher in the whole cell lysate from MSC-H cells than MSC-C cells. The MSC-H cells expressed HMGB1 with flag tag which was generated by viral transfection. The data shown were representative of three repeated tests for each group. b The level of cytosolic HMGB1 was nearly four-fold higher in MSC-H cells than MSC-C cells. But the HMGB1 levels in the nucleus were almost consistent between them. Tubulin and Lamin B1 served as the quality control for cytosolic and nuclear fractionation. The data shown were representative of three repeated tests for each group. c The production of extracellular HMGB1 was at low levels for both MSC-H and MSC-C cells after culture for 12 h. However, MSC-H cells generated a greater amount of extracellular HMGB1, approximately six-fold higher than MSC-C cells after culture for 24 h and 48 h. The difference of LDH release among the groups was not statistically significant, suggesting the increase of extracellular HMGB1 was not due to cell death. The assays were repeated three times for each group. Group comparisons were made by using Student’s t test. *P < 0.05. d The number of migrated MSC-H cells was significantly reduced by HMGB1 antibody (HMGB1 Ab). The assays were repeated three times for each group. Group comparisons were made by using Student’s t test. *P < 0.05. Scale bar = 50 μm. e In the EC-MSC coculture system, the population of CD31-positive cells was significantly decreased at day 14 after the MSC-H cells were treated with HMGB1 Ab. The images were representative of three repeated tests for each group. Scale bar = 50 μm