Fig. 7.

FICZ upregulates OVOL1 expression and mitigates the TGF-β pathway, EMT, cell migration, and extravasation. a RT-qPCR detection of OVOL1 expression in MCF10A-M2 cells stimulated with 6-Formylindolo(3,2-b)carbazole (FICZ; 5 μM) for indicated time points. Statistical analyses were carried out between 0 h group and groups at indicated time points. The results are expressed as mean ± SD. *0.01 < P < 0.05, ***0.0001 < P < 0.001. b Western blotting quantification of OVOL1 expression in MCF10A-M2 cells stimulated with FICZ (5 μM) for indicated time points. Vinculin levels were analyzed to control for equal loading. c Western blotting measurement of the phosphorylation of SMAD2 (p-SMAD2) and OVOL1 expression in MCF10A-M2 cells with inducible OVOL1 knockdown by shRNA #1. Cells were stimulated without or with Doxycycline (Dox) for 2 days, followed by FICZ (5 μM) treatment in serum starvation overnight before adding TGF-β (1 ng/ml) for another 2 h. The vehicle control DMSO was included for FICZ. Vinculin levels were analyzed to control for equal loading. d Western blotting measurement of the expression of TβRI in MCF10A-M2 cells upon OVOL1 knockdown induced by Doxycycline (Dox). Cells were either not treated or treated with Doxycycline (Dox) for 2 days followed by the stimulation of FICZ (5 μM) overnight. Cycloheximide (CHX; 50 µg/ml) was then added to the medium for indicated time points. Quantification of the relative protein level of TβRI is shown in the lower panel. To control for equal loading Vinculin levels were analyzed. The results are expressed as mean ± SD. Statistical analyses were performed using one-way ANOVA. *0.01 < P < 0.05, **0.001 < P < 0.01. e Western blotting analysis of whole-cell lysates (Input) and immunoprecipitants derived from MCF10A-M2 cells stably expressing HA-Ubiquitin (HA-Ub) without or with expressing the OVOL1 targeting shRNA #1. Cells were treated without or with Doxycycline (Dox) for 2 days and FICZ (5 μM) overnight. Total ubiquitination of SMAD7 was probed. f Western blotting analysis of mesenchymal markers expression in MCF10A-M2 cells with inducible OVOL1 knockdown by shRNA #1. Cells were stimulated without or with Doxycycline (Dox) for 2 days, followed by FICZ (5 μM) treatment for 8 h before adding TGF-β (5 ng/ml) for 2 days. The vehicle control DMSO was included for FICZ. g IncuCyte real-time chemotaxis assay for evaluating the migration of MCF10A-M2 cells with inducible OVOL1 knockdown by shRNA #1. Cells were pre-treated without or with Doxycycline (Dox) for 2 days before being seeded into the inserts, followed by FICZ (5 μM) treatment. The vehicle control DMSO was included for FICZ. The results are expressed as mean ± SD. *0.01 < P < 0.05, *** 0.0001 < P < 0.001. h In vivo zebrafish embryo xenograft extravasation experiments of MCF10A-M2 cells without or with the knockdown of OVOL1. MQ water or Dox (to enable induction of the shRNA targeting OVOL1) and DMSO or FICZ (1 μM) was added to the egg water from the first day post injection. Representative images are shown in the left panel. Analysis of the extravasated cell clusters in indicated groups is shown in the right panel. The results are expressed as mean ± SD. ***0.0001 < P < 0.001. i Schematic model of balancing EMP of breast cells by TGF-β, BMP, and downstream transcription factors OVOL1 as well as SNAIL1/2