Fig 1: 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 2: Downstream effectors of SOX11 signalling identified in a mouse model of DCIS that recapitulates human disease. (A) Quantitative polymerase chain reaction (qPCR) analysis of SOX11 and potential SOX11 downstream effectors, i.e. FHAD1, HORMAD1, and TFAP2B, in tumour samples; P < 0.0001. Results are expressed as fold change. Error bars represent standard deviation. (B) ALDH1A1 staining of DCIS lesions that form after mammary intraductal injections of DCIS‐LacZ control and DCIS‐SOX11 cells. Scale bar: 100 μm. (C) ALDH1A1 staining of tumours that formed after mammary fat pad injections of DCIS‐LacZ control and DCIS‐SOX11 cells. Scale bar: 100 μm.
Fig 3: 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 4: Effect of SOX11 expression on DCIS cells. (A) Spheroids from DCIS‐LacZ and DCIS‐SOX11 cells at day 14 grown in BME (top images): relative growth curves showing area (μm2), volume (μm3) and perimeter (μm) of DCIS‐SOX11 spheroids as compared with DCIS‐LacZ control spheroids. Scale bar: 200 μm. (B) CellTiter‐Glo assay results of DCIS‐LacZ control and DCIS‐SOX11 cells. Experiments were performed three times (n = 56 in each sample), and Student's t‐test was 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 < 0.0001 for the three time points. (C) Representative images of cleaved caspase‐3 activity in spheroids from DCIS‐LacZ control and DCIS‐SOX11 cells 8 days after spheres had formed. Scale bar: 200 μm. (D) Relative cleaved caspase‐3 activity detected in spheroids from DCIS‐SOX11 cells as compared with spheroids from DCIS‐LacZ cells at day 8 [Counts fluorescence units (CFU)]. (E) ALDH activity in DCIS‐control versus DCIS‐SOX11 populations. (F) Frequency of CD44+/CD24– cells in DCIS‐SOX11 as compared with DCIS‐control populations. (G) Frequency of CD44+/CD24–/ALDH+ cells in DCIS‐SOX11 as compared with DCIS‐control populations. (H) Frequency of EpCAM–/CD49f+/ALDH+ cells in DCIS‐SOX11 as compared with DCIS‐control populations. All error bars represent standard deviation.
Fig 5: 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).
Supplier Page from DNASU for SOX11 (Homo sapiens) in pENTR223.1 (Gateway donor/master vector)