Figure 7. Kinome-scale siRNA screening indicated that SRC\MEK5 promotes the phosphorylation of BMK1 in respons to CIBM.

(a,b) Human siGENOME siRNA library containing 779 kinases (Supplementary Table S4) was used for the screening. After transfected with siRNA for 48 hrs, resultant A375-P and SK-MEL-28-P drug resistant cells were harvested and analyzed by western blotting as noted. (c) After transfected with control or siSRC for 48 hrs, resultant A375-P and SK-MEL-28-P drug resistant cells were harvested and analyzed by western blotting as noted. (d) A375-P and SK-MEL-28-P cells were treated with/without 2 μM Dasatinib for 1 hr. Resultant cells were harvested and analyzed by western blotting. (e) A375 and SK-MEL-28 cells were serum starved overnight followed by treatment with 2 μM Vemurafenib + 10 nM Trametinib, or/and 2 μM Dasatinib for 1 hr as noted. Then cells were stimulated with serum for 1.5 hrs and phosphorylated BMK1 was detected by mobility retardation²⁷. Resultant cell lysates were analyzed by western blotting using the antibody as noted. (f) A375-MEK5D and SK-MEL-28-MEK5D cells expressing MEK5D were treated with/without 2 μM Dasatinib or 2 μM XMD8-92 for 1 hr. Resultant cells were harvested and analyzed by western blotting as noted. (g) A375, Mel888 and SK-MEL-28 cell combined GI₅₀ of Vemurafenib and Trametinib in the presence or absence of 2 μM XMD8-92 and/or 2 μM Dasatinib as noted. (h) A375 cells were suspended in DMEM and injected subcutaneously into the right flank of 6-week-old Nod/Scid mice. And these tumor-bearing mice were randomized into groups. Mice were injected IP with Vemurafenib (50 mg/kg) (once a day), Trametinib (1 mg/kg) (once a day), XMD8-92 (50 mg/kg) (twice a day), and/or Dasatinib (20 mg/kg) (once a day) as noted. Tumor size was measured twice a week and tumor volume was calculated by using the formula: 0.52 X L X W², where L was the longest diameter and W was the shortest diameter. (i) Scheme for BMK1 enhancing drug resistance to CIBM.