GCS-100 treatment is associated with modulation of cell-signaling proteins and inhibits IGF-1 and TNF-α pathway stimulation. (A-B) RPMI 8226 cells were exposed to 500 μg/mL GCS-100 for up to 48 hours and after preparation of whole-cell lysates examined by Western blot. A total of 50 μg of protein was separated using 12% SDS-PAGE, transferred to PVDF membrane, and probed using the indicated antibodies. (C-E) RPMI 8226 cells were incubated in serum-free media for 1 hour and then cultured with 500 μg/mL GCS-100 or control media for 2 hours. Cells were then stimulated with TNF-α (5 ng/mL), IGF-1 (100 ng/mL), or IL-6 (10 ng/mL) for 30 minutes. Whole-cell lysates were prepared and 50 μg of protein resolved using 12% gel, transferred to PVDF, and probed with the indicated antibodies. β-Actin was used as a loading control.