Fig. 5.

OVOL1 interacts with and stabilizes SMAD7, thereby enhancing the degradation of TβRI. a Quantification of TGF-β type I receptor (TβRI) protein (left) or TBRI mRNA expression (right) by western blotting or RT-qPCR, respectively, in MDA-MB-231 cells with inducible OVOL1 ectopic expression. Cells were kept in the presence or absence of Doxycycline (Dox) for 2 days. To control for equal loading, GAPDH levels were analyzed. NS not significant. b TβRI expression quantified by western blotting in MDA-MB-231 cells with OVOL1 ectopic expression induced by Doxycycline (Dox; left). Cells were cultured in the presence or absence of Dox for 2 days followed by the stimulation of cycloheximide (CHX; 50 µg/ml) for indicated time points. Quantification of the relative protein level of TβRI is shown in the right panel. Statistical analyses were performed at the indicated time points. To control for equal loading Vinculin levels were analyzed. The results are expressed as mean ± SD. *0.01 < P < 0.05. c Western blotting detection of TβRI expression in MDA-MB-231 cells with the inducible expression of OVOL1. Cells were kept in the presence or absence of Doxycycline (Dox) for 2 days followed by the treatment of proteasome inhibitor MG132 (5 μM) or lysosome inhibitor BafA1 (20 nM) for 6 h. d Western blotting quantification of whole-cell lysates (Input) and immunoprecipitants derived from HEK293T cells with inducible OVOL1 expression. Cells were either not stimulated or stimulated with Doxycycline (Dox) for 1 day and then transfected with HA-Ub and constitutively active TβRI-FLAG (caTβRI-FLAG). e SMAD7 expression measured by RT-qPCR in MDA-MB-231 cells with inducible expression of OVOL1. Cells were either not treated or treated with Doxycycline (Dox) for 2 days. The results are expressed as mean ± SD. **0.001 < P < 0.01. f Western blotting detection of whole-cell lysates (Input) and immunoprecipitants derived from HEK293T cells transfected with indicated FLAG-SMADs and OVOL1. g Western blotting quantification of 5% cell lysates (Input) and analysis of OVOL1 and SMAD7 immunoprecipitants derived from MCF10A-M2 cells. The SMAD7 antibody was added to the cell lysates to pull down SMAD7 and the IgG isotype was included as a control. h Western blotting measurement of the expression of HA-SMAD7 in HEK293T cells with inducible OVOL1 ectopic expression (upper). Cells were either not treated or treated with Doxycycline (Dox) for 2 days followed by the stimulation of cycloheximide (CHX; 50 ug/ml) for indicated time points. Quantification of the relative protein level of HA-SMAD7 is shown in the lower panel. Statistical analyses were performed at the indicated time points. To control for equal loading Vinculin levels were analyzed. The results are expressed as mean ± SD. *0.01 < P < 0.05, **0.001 < P < 0.01. i Western blotting analysis of whole-cell lysates (Input) and immunoprecipitants derived from MDA-MB-231 (left) or MCF10A-M2 cells (right) stably expressing HA-Ubiquitin (HA-Ub) without or with ectopic expression of OVOL1 (left) or expression the OVOL1 targeting shRNA #1 (middle). Cells were treated without or with Doxycycline (Dox) for 2 days. Total ubiquitination of SMAD7 was probed. Quantification of the relative protein level of HA-Ubiquitin (HA-Ub) is shown in the right panel. The results are expressed as mean ± SD. *0.01 < P < 0.05, **0.001 < P < 0.01. j Schematic working model indicating the action of OVOL1 on SMAD7 and TβRI regulation