Figure 4.

The p38/MAP Kinase and NFκB pathways are activated in sunitinib resistant cells. (A) A Venn diagram of genes up- or down-regulated in sensitive or resistant sunitinib cells. (B) Heatmaps of the most up-regulated pathways (Autophagy, Lysosome, Cytokines, NFκB and ROS) for untreated 786-O cells (C1, C2) or after incubation for 48 hours with sunitinib 2.5μmol/L (S1, S2) and in sunitinib resistant cells (R1, R2). (C) Determination of ROS production in cells treated with 2.5 μmol/L sunitinib for 48 hours in sunitinib sensitive (S) and resistant (R) cells. H₂O₂ serves as a positive control of ROS production. (D) A reporter gene containing three NFκB binding sites was transfected into either untreated (C) or sunitinib (sun, 2.5 μmol/L) treated 786-O cells and sunitinib resistant 786-O cells. The percentages or normalized luciferase counts were measured for 48 hours post transfection. (E) The total (p38) and phosphorylated (pp38) forms of p38 MAP Kinase were detected by immuno-blotting in naïve (C) or sunitinib treated for 24 hours (sun, 2.5 μmol/L) 786-O cells and sunitinib resistant (R) 786-O cells. HSP60 is shown as a loading control.