Fig. 6

Insulin-stimulated CXCL12/CXCR4 axis promotes the cytoskeleton reorganization and migration in BCAHC-1 cells. A FITC-phalloidin staining of BCAHC-1 cells treated for 8 h with vehicle or 10 nM insulin alone or in combination with 10 μM CXCR4 antagonist AMD3100 or 2 mM metformin that was added to culture medium 18 h before the treatments. Cells were then cultured for additional 8 h to conditioned medium (CM) collected from CAFs, which were previously treated for 8 h with vehicle or 10 nM insulin, as indicated. Cells were stained with FITC-phalloidin to detect F-actin stress fibers (green) and with DAPI to detect nuclei (blue). B Fluorescence intensities of the number of stress fibers/cell was quantified based F-actin staining in 10 random fields for each treatment; results are shown as fold change of relative fluorescence units (RFU). Scale bar: 25 μm. Values represent the mean ± SD of three independent experiments performed in triplicate. (*) indicates p < 0.05 for cells exposed to treatments versus vehicle. C Transwell migration assay in BCAHC-1 cells treated for 8 h with vehicle or 10 nM insulin alone or in combination with 10 μM CXCR4 antagonist AMD3100 or 2 mM metformin that was added to culture medium 18 h before the treatments. Cells were then cultured for additional 8 h to conditioned medium (CM) collected from CAFs, which were previously treated for 8 h with vehicle or 10 nM insulin, as indicated. D Evaluation of cell migration in 10 random fields in each of three independent experiments performed in triplicate. Scale bar: 200 μm. (*) indicates significant differences with respect to BCAHC-1 (+vehicle) exposed to CM/CAFs (+vehicle) sample (p < 0.05); (black square) indicates significant differences with respect to BCAHC-1 (+Insulin) exposed to CM/CAFs (+vehicle) sample (p < 0.05); (black circle) indicates significant differences with respect to BCAHC-1 (+vehicle) exposed to CM/CAFs (+Insulin) sample (p < 0.05); (white circle) indicates significant differences with respect to BCAHC-1 (+Insulin) exposed to CM/CAFs (+Insulin) sample (p < 0.05)