Fig. 1

The effects of rHMGB1 on breast cancer migration and invasion. Chemo-migration was tested in a Fluoroblok™ Transwell migration assays for MDA-MB-231 P and MDA-MB-231 BM with 100 ng/ml rHMGB1 in 1% BSA DMEM as the chemoattractant. Representative images of cells which successfully migrated to the lower surface of the membrane were visualized using an inverted fluorescence microscope are shown (a). Bar graphs that represent migration of control (1% BSA DMEM) calculated from the number of migrated cells of 10 images taken from 2 wells per conditions (b). Cell invasion was tested in a Matrigel-coated Transwell invasion assay for MDA-MB-231 P and MDA-MB-231 BM with 100 ng/ml rHMGB1 in 1% BSA DMEM for 48 h. Representative images of cells that successfully invaded the lower surface of the insert visualized using an inverted fluorescence microscope are shown (c). Bar graphs represent percent invasion of controls unstimulated (1% BSA DMEM) calculated from the number of invaded cells of 10 images taken from 2 wells as per conditions (d). Results are presented as mean ± SD of duplicate independent experiments. Scale bar = 100 μm and original magnification 100X. For 3-D tumor spheroid-based migration assay. Tumor spheroids of MDA-MB-231 P and MDA-MB-231 BM were transferred to each well of a 96-well flat-bottomed plate coated with 0.1% gelatin which contained rHMGB1 at various concentrations in 0.1% BSA DMEM. Representative bright-field images of cell migration of MDA-MB-231 P at 48 h (e and f) and MDA-MB-231 BM at 96 h (g and h) are shown. Bar graphs represent percent cell migration normalized against the control untreated cells. Results are presented as mean ± SD of duplicate independent experiments. *P < 0.05