Fig. 1.

Hypoxia activates the CDCP1-Src pathway. (A) HCT116 cells were exposed to 21% O₂ (normoxia; N) or 1% O₂ (hypoxia; H) for 12, 18, 24, 36, or 64 h. The anti-CDCP1 antibody was used for immunoprecipitation (IP) and immunoblotting. An anti-phosphotyrosine antibody was used for immunoblotting. (B) HCT116 cells were exposed to N or H for 48 h. Lysates were immunoblotted for the phosphorylation of CDCP1 and Src-family kinases using a specific CDCP1 P-734 antibody that recognizes CDCP1 when phosphorylated on Tyr734 and a P-416 SFK antibody that recognizes the activation loop of Src-family kinases when phosphorylated at Tyr416. An anti-SFK antibody was used as loading control. (C) DLD1 and HCT116 cells cultured in 0.1% or 10% FBS were treated ±100 μM DFO and/or 50 nM dasatinib for 24 h. The anti-CDCP1 antibody was used for immunoprecipitation and immunoblotting. An anti-phosphotyrosine antibody was used for immunoblotting. (D) Two retroviral shRNAs targeted against CDCP1 were used to stably knock down CDCP1 in MCF10A cells. An anti-tubulin antibody was used as loading control. Stable cell lines were exposed to N or H for 48 h. Lysates were immunoblotted for the phosphorylation of CDCP1 and SFKs as in B using the specific CDCP1 P-734 antibody and the anti-SFK antibody for loading control. (E) Stable CDCP1 knockdown MCF10A cell lines were serum-starved and exposed to 1% O₂ for 24 h; subsequently, the cells were seeded in transwells and returned to 1% O₂ for 24 h. Cells were fixed and stained with crystal violet (0.1%; Lower). The number of cells that migrated to the bottom of the filter was counted and the data are reported as fold induction over the pRenilla control cells. The data presented are the result of triplicate analyses and the error bars indicate SEM. *P = 0.088, ***P = 0.0007.