Fig 1: Willin/FRMD6 over-expression results in a general reversal of the findings observed for knock-down cells and activate the mechanical activity of SH-SY5Y cells. (A) Quantitative Western blot analysis of Willin/FRMD6 for control (Vector) and Willin/FRMD6 overexpressing (Willin) SH-SY5Y cells. Means and SEM (error bars) were calculated from two independent experiments, each of which was conducted in triplicates. ß-actin serves as a loading control of the lysates. (B) qPCR analysis of Willin/FRMD6 expression in Vector and Willin cells. Means and SEM (error bars) were calculated from two independent experiments, each of which was conducted in triplicates. (C) Growth curve of Vector and Willin cells. Means and SEM (error bars) were calculated from three independent experiments, each of which was conducted in triplicates. (D) Assessment of migration of shScr and shWillin cells in Boyden chambers after 24 h. Means (horizontal lines) and SEM (error bars) were calculated from two independent experiments, each of which was conducted in triplicates. (E) Representative phase-contrast images (upper row), immunostainings against F-actin (red), vinculin (green) and DNA (blue; middle row), and TAZ (lower row) of Vector and Willin SH-SY5Y cells. Scale bars: 30 µm. Comparison of (F) cell area, (G) cell elongation, (H) nuclear/cytosolic TAZ ratio, and (I) volume by which cells indent into the ERISM substrate for Vector and Willin cells. Each data point represents the value measured for one cell. Lines indicate means, error bars SEM. Groups were compared using Student’s t-test; ns: p > 0.05; *p = 0.05; **p = 0.01; ***p = 0.001.
Fig 2: Expression of YAP1 and TAZ proteins in postnatal pituitaries. Immunofluorescence using specific antibodies on frontal sections of dissected postnatal pituitaries at P21. (A) Immunofluorescence using antibodies against the pituitary stem cell marker SOX2 (red, arrowheads) showing positive staining in the periluminal epithelium and in small groups of cells throughout the anterior lobe parenchyma. Antibodies against endomucin mark epithelial cells surrounding blood vessels (green, arrows). (B–D) Immunofluorescence staining against total TAZ protein (B), total YAP1 protein (C) and phosphorylated YAP1 (S127) protein (D) in red. All three are detected in periluminal cells (white arrowheads) as well as structures resembling blood vessels (white arrows). (E–G) Double immunofluorescence staining on pituitary sections from Sox2Egfp∕+ animals using antibodies against GFP protein in green detecting EGFP and marking SOX2+ cells together with antibodies against TAZ protein (E), total YAP1 protein (F) and phosphorylated YAP1 (S127) protein (G) in red. Arrows in (E,F) indicate examples of cells with nuclear TAZ or YAP1 protein. Yellow arrowheads in G note examples of GFP+ cells with strong nucleocytoplasmic pYAP1 protein localization and green arrowheads indicate GFP+ cells in lower pYAP1 levels in the nucleus. Nuclei are counterstained with DAPI (blue). Abbreviations: pl, posterior lobe; al, anterior lobe; il, intermediate lobe. Axes in (A) applicable to all panels: d, dorsal; v, ventral; ri, right; le, left. Scale bars 100 μm in (A–D), 20 μm in inserts and 50 μm in (E–G).
Fig 3: Effect of TAZ or YAP knockdown plus and minus TGFß on cell proliferation of canine OSA cell lines. Representative immunofluorescence images of the mitotic marker phosphor-histone H3 (pHH3) taken at 20X objective lens for primary tumour-derived cell lines (a), and metastatic cell lines (b). Graphs depict the relative percentage of pHH3 positivity cells, as determined by dividing the number of pHH3 cells by the number of cells in the field of view. Five images were taken per experimental group and averaged. Error bars depict average ± SEM from three independent experiments. Scale bar = 100 µm. Asterisks depict statistical differences as determined by a two-way ANOVA with post-hoc Tukey-Kramer, * indicates p < 0.05, ** indicates p < 0.010, *** indicates p < 0.0010
Fig 4: MOB1B and YAP/TAZ pathway plays an important role in PINK1-dependent mitophagy-mediated inhibition of myeloma cell migration. A) Higher expression of MOB1B demonstrated a trend for better overall survival in Arkansas myeloma microarray dataset. B,C) Kaplan–Meyer analysis of overall survival in the Arkansas and Mulligan datasets basing on the expression of B) YAP or C) TAZ in CD138+ cells of myeloma patients. Survival analysis was performed using a log-rank test. D,E) MOB1B specific shRNA knockdown restored YAP/TAZ expression. D) PINK1 overexpressing MM.1S cells and E) PINK1 overexpressing MM.1R cells were transduced with two different MOB1B shRNA. MOB1B, YAP, and TAZ expression was measured by Western blot analysis. F) MOB1B specific shRNA knockdown did not affect myeloma cell proliferation. MM.1S cells (left panel) and MM.1R cells (right panel) were transduced with two different MOB1B shRNA and cell proliferation was measured by MTT assay. G) Overexpression of YAP or TAZ did not affect myeloma cell proliferation. PINK1 overexpressing MM.1S cells (left panel) and PINK1 overexpressing MM.1R cells (right panel) were transduced with YAP overexpressing vector or TAZ overexpressing vector. Cell proliferation was measured by MTT assay. H) MOB1B specific shRNA knockdown restored myeloma cell transwell migration that was inhibited by PINK1 overexpression. PINK1 overexpressing MM.1S cells (left panel) and PINK1 overexpressing MM.1R cells (right panel) were transduced with two different MOB1B shRNA. Myeloma cell migration was measured by transwell assay. I) Overexpression of YAP or TAZ restored myeloma cell transwell migration that was inhibited by PINK1 overexpression. PINK1 overexpressing MM.1S cells (left panel) and PINK1 overexpressing MM.1R cells (right panel) were transduced with YAP overexpressing vector or TAZ overexpressing vector. Myeloma cell migration was measured by transwell assay.
Fig 5: Expression of KRAS G12V alone or in combination with p16 loss and small-T-antigen expression in U57810 cells does not change Yap/Taz transcriptional activity. (A) Representative Western blots showing total Yap, Taz, and phosphorylated Yap levels in U57810 cells, with Gapdh as loading control. (B) Effect of KRAS G12V expression on 8xGTIIC Yap/Taz Tead1-4 luciferase reporter normalised to Tk-renilla in U57810 cells. Data is presented as fold change compared to control (mean ± SD) from independent experiments. (C) Representative Western blots showing total Yap, Taz, and phosphorylated Yap S112/Taz S89 levels in U57810 cells, with Gapdh as a loading control. (D) Effect of KRAS G12V expression combined with p16 loss and small-T-antigen expression on 8xGTIIC Yap/Taz Tead1-4 luciferase reporter normalised to Tk-renilla in U57810 cells. Data is presented as fold change compared to control (mean ± SD) from 3 independent experiments.
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