Fig 2: SMOC2 interacts with integrin ß3 to mediate EMT.a ACHN and b 786-O cells were transfected with either scrambled siRNA (ssiRNA) or integrin ß3 siRNA (siITB3) 24 h prior to 5 ng/mL SMOC2 recombinant protein treatment, then protein harvested after 24 and 48 h. Western blot was performed on whole cell extracts for integrin ß3 (ITGB3), and EMT markers fibronectin and aSMA. GAPDH immunoblotting served as a loading control. Images are representative of n = 3; *P < 0.05 determined by t-test.

Fig 3: SMOC2 overexpression induces EMT markers in RCC cells.a ACHN and b 786-O cells were transfected with either a SMOC2-Myc or empty-Myc vector, then protein harvested at indicated times. Cell extracts were subjected to Western blot analysis for fibronectin, E-cadherin, aSMA, vimentin and SMOC2 (Myc). GAPDH immunoblotting served as a loading control. Representative of n = 3; *P < 0.05 determined by t-test.

Fig 4: SMOC2 binds to integrin ß3.a ACHN and b 786-O cells were transfected with either a SMOC2-Myc or empty-Myc vector, then protein harvested after 24 h. Cell extracts were immunoprecipitated for Myc. Western blot analysis was performed on whole cell extracts (2.5% Input), supernatant and Myc-immunoprecipitated samples for Myc, integrin ß3 (ITGB3) and GAPDH. Western blot images are representative of repeated experiments.

Fig 5: SMOC2 affects the morphology and colony organization of epithelial cells.ACHN cells were transfected for 24 h with a an empty-Myc or SMOC2-Myc vector, and b silencing RNA (ssiRNA) or SMOC2 siRNA, then evaluated for their morphology. siRNA transfections were cotreated with TGFß1. Cells were outlined in red for increased visibility. c After 24 h of transfection with an empty-Myc or SMOC2-Myc vector, MDCK cells formed a confluent monolayer then scratched for a Scratch assay. Images of MDCK cells were taken 24 h after the inflicted scratch. d 786-O cells were transduced with a SMOC2-luc or empty-luc vector, which were used to form a confluent monolayer, then scratched for a Scratch assay. Live cell analysis was performed on such 786-O cells over a 43 h period to calculate the percentage of wound confluence as a function of time for triplicates (15 000 cells/well). Wound confluence (%) represents the fractional area of the wound that is occupied by cells. a, b were taken at 20X magnification and c was taken at 10X magnification. Scale bar: 50 µm. Images are representative of n = 3; *P < 0.05 determined by t-test between SMOC2 and control cells at each time point.