Fig 1: SOX11 increases the invasive activity of DCIS cells in vitro and promotes the growth and progression of DCIS cells in vivo. (A) Representative images of DCIS-LacZ control and DCIS-SOX11 spheroid invasion in collagen I at day 2. Scale bar: 500 µm. Error bars represent standard deviation. (B) Area of invasion of DCIS-SOX11 as compared with DCIS-LacZ control spheroids in collagen I at day 2. P = 0.0162. Experiments were performed three times. (C) Representative images of in situ and microinvasive lesions formed from DCIS-LacZ control cells that appeared in mice injected intraductally 6–7 weeks after xenografting. Lamin A/C stain detects human cells. Scale bar: 100 µm. (D) Representative images of in situ lesions, microinvasive lesions and invasive lesions formed from DCIS-SOX11 cells that appeared in mice injected intraductally 6–7 weeks after xenografting. Lamin A/C stain detects human cells. Scale bar: 100 µm. (E) Results from intraductal injections of DCIS-LacZ control and DCIS-SOX11 cells; representative images and quantification of in vivo bioluminescence 6–7 weeks after injection of DCIS-LacZ control and DCIS-SOX11 cells. Results are expressed in photons per second (p/s); P < 0.0001. Each dot represents the total photon count from each injected mammary gland. Error bars represent standard error of the mean (SEM).
Fig 2: Apoptosis of AT2 cells in each group. (A-H) Flow cytometric analysis of cell apoptosis; (I) Quantification of cell apoptosis. The graph shows the percentage of late apoptotic cells (upper right quadarant in plots). CS significantly increased the apoptosis of AT2 cells. This increase in apoptosis was attenuated by SOX11 overexpression, and aggravated by SOX11 knockdown. In addition, FAK antagonism inhibited the effects of SOX11 overexpression. Data are presented as the means ± standard error of the mean. **P<0.01. AT2, alveolar type II; CS, cell stretch; SOX, Sex-determining gene on the Y chromosome related high mobility group box; FAK, focal adhesion kinase.
Fig 3: Effect of SOX11 expression on postnatal mammary epithelial cell growth, morphogenesis, and clonogenicity. (A) CellTiter-Glo assay results for MCF10A-LacZ-control and MCF10A-SOX11 cells. Experiments were performed three times (n = 18 in each sample), and anova and multiple comparisons were used for statistical analysis. The values obtained in each time point [counts per second (CPS)] were normalized by dividing by the value obtained at day 0 in each population. P < 0.05 for MCF10A-SOX11 versus MCF10A-LacZ; P < 0.0001 for MCF10A-SOX11 versus MCF10A-luc. (B) Representative images of MCF10A-LacZ control and MCF10A-SOX11 mammospheres that were grown from spheroids formed in low-attachment plates, 10 and 14 days after addition of BME. Experiments were performed three times. Scale bar: 200 µm. (C) Quantification of clonogenicity and mammosphere-initiating capacity. Left: percentage of MCF10A cell populations plated in two-dimensional (2D) culture that form colonies. Centre: percentage of colonies with basal or myoepithelial morphology of the total cell number plated in 2D culture. Right: percentage of mammospheres formed from cells in 3D culture. All experiments were performed three times. ***P < 0.001, ***P < 0.0001. (D) Typical morphologies observed for colonies of MCF10A-LacZ and MCF10A-SOX11 cells in (C) (left) (scale bar: 1 mm) and for mammospheres derived from single MCF10A-LacZ and MCF10A-SOX11 cells embedded in methylcellulose (right). Single cells proliferated and formed cell clusters with a large central lumen in MCF10A-SOX11 mammospheres. Scale bar: 200 µm. (E) Quantification of cleaved caspase-3 levels (left) and representative images of MCF10A-luc, MCF10A-LacZ and MCF10A-SOX11 spheroids 5 days after sphere formation. The experiment was performed three times; P < 0.05. Scale bar: 200 µm. All error bars represent standard deviation. CFU, Counts fluorescence units.
Fig 4: SOX11, an embryonic mammary epithelial marker, predicts poor clinical outcome in breast cancer patients and is expressed in preinvasive breast lesions. (A) Disease-free survival (DSS) curves for 1032 breast cancer patients with lymph node-negative disease and with low and high SOX11 expression from analysis of microarray data from the METABRIC dataset. Expression data were stratified into quartiles based on SOX11 expression, and the lowest expression quartile (Q1) was treated as the baseline for the subsequent pairwise comparisons with the remaining quartiles. The statistical significance of pairwise comparisons was assessed with the Wald test. (B) Overall survival (OS) curves for 1032 breast cancer patients with lymph node-negative disease and with low and high SOX11 expression from analysis of microarray data from the METABRIC dataset. Expression data were stratified into quartiles based on SOX11 expression, and the lowest expression quartile (Q1) was treated as the baseline for the subsequent pairwise comparisons with the remaining quartiles. The statistical significance of pairwise comparisons was assessed with the Wald test. (C and D) Haematoxylin and eosin stain (C) and SOX11 expression (D) in DCIS lesions. Scale bar: 200 µm. HR, hazard ratio.
Fig 5: (A) Reverse transcription-quantitative PCR and (B) western blot analysis showing the downregulation of SOX11 and FAK at the mRNA and protein level in AT2 epithelial cells following CS. Data are presented as the mean ± standard error of the mean. *P<0.05. AT2, alveolar type II; CS, cell stretch; SOX, Sex-determining gene on the Y chromosome related high mobility group box; FAK, focal adhesion kinase.
Supplier Page from Abcam for Anti-SOX11 antibody [EPR8191(2)]