Fig. 5

Insulin triggers CXCL12 expression in cancer-associated fibroblasts (CAFs). mRNA (A) and protein (B) expression of CXCL12 evaluated respectively by real-time PCR and immunoblotting in CAFs exposed to vehicle (−) or 10 nM insulin (Ins), as indicated. In RNA experiments, values were normalized to actin beta (ACTB) expression and shown as fold changes of mRNA expression upon treatments respect to vehicle. C CXCL12 expression evaluated by immunofluorescence assays in CAFs treated with vehicle or 10 nM insulin for 8 h. Nuclei were stained by DAPI (blue signal). Fluorescence intensities were quantified in 10 random fields for each condition and results are expressed as fold change of relative fluorescence units (RFU) over cells treated with vehicle (set as one-fold change). Scale bar: 25 μm. D Assessment of CXCL12 protein levels in conditioned medium (CM) collected from CAFs treated for 8 h with vehicle (−) and 10 nM Ins. Ponceau red staining was used as a loading control for CM. Side panels show densitometric analysis of the blots normalized to the respective loading control. Results shown are representative of at least three independent experiments. (*) indicates significant differences with respect to vehicle (−) sample (p < 0.05)