Figure 7

TSC^−/−A⁺ cells release IGF-1 in culture medium. The medium was removed and the cells washed once, and then fresh medium was added and collected 24 h later for IGF-1 assay. The collected media were immediately stored at −80°C until IGF-1 determination by ELISA. IGF-1 release is shown as a percentage of controls (fixed at 100%). A: The cells were cultured for the indicated times (days) in standard medium. IGF-1 release was quite constant during the first week in culture, and then progressively increased to peak levels. B: The cells were grown for five days, and incubated for 24 h with fresh medium (control, C) with and without the presence of cycloheximide (CHX, 10 µg/mL) or rapamycin (Rapa, 5 ng/mL). CHX greatly reduced IGF-1 release, whereas rapamycin did not change it. C: The cells were grown for five days in standard medium (control, C) with and without the presence of anti-EGFR (clone 225, 5 µg/mL) or anti-IGF-1R antibody (αIR3, 5 µg/mL). Both antibodies greatly enhanced IGF-1 release, but anti-IGF-1R was significantly more effective than anti-EGFR. Mean values ± SEM; between-group differences evaluated by ANOVA test: *** P< 0.001 vs control; ^○○○ P< 0.001 vs 5 days; § P< 0.05 vs 14 days; € P < 0.05 vs anti-EGFR treatment.