Specificity of MGF inhibitors and IGF-1 production by HMCLs. (A) XG-2 cells were starved at 37°C for 18 hours in Syn H serum-free culture medium. Cells were then cultured without cytokine (control) or with either IL-6 (200 pg/mL) or IGF-1 (100 ng/mL) or HGF (20 ng/mL) and without inhibitor or with anti–IL-6 mAb (10 μg/mL) or IGF-1R inhibitor (1 μM) or anti-HGF mAb (25 μg/mL) for 20 minutes at 37°C in the Syn H culture medium. The receptor kinase inhibitors were added to the cells for 4 hours at the end of starvation culture and during exposure to rMGF. The anti-MGF antibodies were preincubated with the rMGF for 1 hour before to be added to cells. Cell lysates were immunoblotted with anti–phospho-Akt antibody and then reprobed with anti-akt antibody, anti–phospho-MAPK antibody, and then reprobed with anti-MAPK, anti–phospho-Stat3 antibody, and then reprobed with anti-Stat3 antibody. Anti–β actin was used as a loading control. (B) HMCLs were cultured for 2 days without cytokine in the Syn H serum-free culture medium. Cell lysates were immunoblotted with an anti–IGF-1 antibody. Anti–β actin was used as a loading control and the U266 HMCL as a negative control for IGF-1 production (no expression of IGF-1 gene using Affymetrix microarrays). (C) XG-2 cells were starved for 2 hours in Syn H serum-free culture medium and then cultured without cytokine (control) or with increased concentrations of rIGF-1 for 4 days. Results are the mean luminescent signals ± SD determined in sextuplicate culture wells and are those of 1 experiment representative of 3. Data are expressed as percentage of the signal obtained without cytokine. The mean value was significantly different from that obtained in the control group using a Student t test (*P ≤ .05).