Fig. 4.

A combination of afatinib and thalidomide but not TMZ induces TNF dependent ERK activation. (A) GBM12 cells were treated with afatinib (1 μM) or TMZ (1 μM) for the indicated times followed by western blotting with phosphorylated ERK, ERK, or β-actin antibodies. (B) GBM12 cells were exposed to indicated drugs for 48 hours followed by western blot. (C–D) Similar experiments to that described in A and B were performed with GBM9 cells. (E) Mice were injected intracranially with GBM12 cells. After 10 days, mice were treated with afatinib or TMZ for 1, 2, or 7 days, followed by euthanasia (at 1, 2, or 7 days after treatment) and tumors were then harvested and western blot was conducted on tumor tissue to detect ERK activation. (F) Mice with GBM12 orthotopic tumors were treated with indicated drugs for 48 hours and brain tumor lysates were analyzed by western blot. (G–H) Similar experiments as performed in E and F in a GBM9 orthotopic model. (I) GBM9 and GBM12 cells were treated with TMZ for 48 hours followed by TNF ELISA. (J) Mice with GBM12 orthotopic tumors were treated with indicated drugs for 48 hours and brain tumor lysates were analyzed by TNF ELISA. (K) A similar experiment was conducted in a GBM9 orthotopic model. (L–M) Time course of TNF upregulation in mice with a GBM12 orthotopic tumor exposed to afatinib or TMZ for 1, 2, and 7 days. (N–O) A similar experiment was performed in a GBM9 orthotopic model. (P–Q) Western blot analysis of BIM protein expression in GBM12 and GBM6 cells treated with indicated drugs for 48 hours. Data are presented as mean ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01; n.s., not significant.