1. DEAR1 blocks TGF-β–induced anoikis resistance, and TGF-β and SMAD3 signal transduction.
DEAR1 blocks TGF-β–induced anoikis resistance, and TGF-β and SMAD3 signal transduction. A, suspension culture of DshR and CshR clones of 76N-E6 HMECs. Cells were seeded into ULA dishes with or without TGF-β treatment (2 ng/mL) for 4 days. Note cellular aggregates in DshR1 and DshR2 cells with and without TGF-β treatment. B, colony formation of suspension cells. After 4 days in ULA suspension culture, cells were plated in regular tissue culture dishes, cultured for 7 days, and colonies stained with crystal violet. C, Western blot analysis of active caspase-3 (cas.-3). Suspension cells were collected at the indicated time points and lysed in 1× SDS sample buffer. D, DEAR1 inhibition of TGF-β response element–driven luciferase activity. HA-tagged DEAR1 and empty vector control were transiently cotransfected with luciferase reporters with TGF-β response elements (CAGA12 or PAI-1) into HEK293T cells. After 24 hours, cells were treated with or without TGF-β (1 ng/mL) for 24 hours and luciferase activity measured. E, DEAR1-mutant shRNA rescue of DEAR1 inhibition of TGF-β signaling. Along with CAGA12 luciferase reporters, empty vectors, DEAR1 (DR1), or rescue-mutant DEAR1 [DR1(mut)] were transiently cotransfected with DshR or CshR into HEK293T cells. After 24 hours, cells were treated with or without TGF-β (1 ng/mL) for 24 hours and luciferase activity was measured. Right, Western blot analysis of same samples used in luciferase assays shown at the left. F, luciferase reporter assay to measure the response of 76N-E6 DshR and CshR clones to TGF-β (1 ng/mL). CAGA12 was transfected in cultured cells. After 24 hours, cells were treated with or without TGF-β (1 ng/mL) for 24 hours and luciferase was measured. The values were normalized to the protein amount. G, the effect of tumor-derived mutation of DEAR1 on TGF-β signal transduction. Various HA/DEAR1 mutants were cotransfected into HEK293T cells with CAGA12 reporter. After 24 hours, cells were treated with or without TGF-β (1 ng/mL) for 24 hours and luciferase activity was measured. H, cotransfection of DshR with DEAR1 specifically reverses DEAR1 inhibition of SMAD3 signaling. Myc/SMAD3 was cotransfected with DEAR1 and DshR or CshR as well as CAGA12 luciferase reporter in HEK293T cells. After 24 hours, cells were treated with or without TGF-β (1 ng/mL) for 24 hours and luciferase was measured.
Nanyue Chen et al. Cancer Discovery 2013;3:
©2013 by American Association for Cancer Research