Supplementary Figure S5 from Deubiquitinase Activity Profiling Identifies UCHL1 As a Candidate Oncoprotein That Promotes TGFβ-Induced Breast Cancer Metastasis

Supplementary Figure S5. Related to Figure 4. A, Real-time scratch assay of MDA-MB-231 cells that are engineered to overexpressed TβRI, SMAD2 or SMAD3 using the IncuCyte system. Western blot analysis of TβRI, SMAD2 or SMAD3 expression levels in MDA-MB-231 cells (above). Representative scratch wound results at the end time point of the experiment are shown (middle). The region of the original scratch is colored in purple and the area of cell migration into the scratch is colored in yellow. Relative wound density (closure) is plotted at indicate times (below). ****, P < 0.0001, two-way analysis of variance (ANOVA). Same blot was used for TβRI and GAPDH (loading control). UCHL1 and SMAD2/3 results were derived from another blot using the same corresponding cell lysates. B, Real-time scratch assay of MDA-MB-436 shUCHL1 cells that are engineered to overexpressed TβRI, SMAD2 or SMAD3 using the IncuCyte system. Western blot of TβRI, SMAD2 or SMAD3 expression levels in MDA-MB-436 shUCHL1 cells (above). Representative scratch wound results at the end time point of the experiment are shown (middle). Relative wound density (closure) is plotted at indicate times (below). **, P < 0.01, ****, P < 0.0001, two-way analysis of variance (ANOVA). Same blot was used for TβRI and GAPDH (loading control). UCHL1 and SMAD2/3 levels in corresponding cell lysates were determined using another blot. C, Real-time scratch assay of MDA-MB-231 cells that overexpress with UCHL1 WT or UCHL1 C90A using the IncuCyte system. Representative scratch wound results at the end time point of the experiment are shown (above). Relative wound density (closure) is plotted at indicate times (below). ***, P < 0.001, ****, P < 0.0001, two-way analysis of variance (ANOVA). D, In vivo zebrafish extravasation assay of MDA-MB-231 cells that overexpress UCHL1 WT or UCHL1 C90A. Representative images of zebrafish tail fin with zoom in of invasive cells (above). Analysis of invasive cell numbers of control, UCHL1 WT and UCHL1 C90A groups in zebrafish extravasation experiment (below). **, P < 0.01, ****, P < 0.0001, two-way analysis of variance (ANOVA).