1. High Expression of Sphingosine 1-Phosphate Receptors, S1P1 and S1P3, Sphingosine Kinase 1, and Extracellular Signal-Regulated Kinase-1/2 Is Associated with Development of Tamoxifen Resistance in Estrogen Receptor-Positive Breast Cancer Patients 
Carol Watson, Jaclyn S. Long, Clare Orange, Claire L. Tannahill, Elizabeth Mallon, Liane M. McGlynn, Susan Pyne, Nigel J. Pyne, Joanne Edwards 
The American Journal of Pathology 
Volume 177, Issue 5, Pages (November 2010)
DOI: /ajpath
Copyright © 2010 American Society for Investigative Pathology Terms and Conditions

2. Figure 1
A: Western blot demonstrating that treatment of MCF-7 cells with SK1 siRNA substantially reduces the expression of SK1 (Mr 42,000) compared with scrambled siRNA-treated MCF-7 cells, and Western blot showing ERK-2 levels do not change in scrambled versus SK1 siRNA-treated cells. B: Graph demonstrating that tamoxifen significantly reduces basal [3H]thymidine incorporation in scrambled and SK1 siRNA-treated cells. Treatment with siRNA SK1 did not significantly reduce basal DNA synthesis compared with scrambled siRNA-treated cells. Treatment with siRNA SK1 increased tamoxifen-inhibited DNA synthesis by 57% versus scrambled siRNA-treated cells.
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2010 American Society for Investigative Pathology Terms and Conditions

3. Figure 2
A: Western blot for SK1 demonstrating that antibodies detect a single band corresponding to 42 kDa, the recognized size of the SK1 protein. B: Cell pellets demonstrating that treatment with siRNA SK1 reduced SK1 expression as assessed by IHC versus scrambled siRNA-treated cells. C: Example of SK1 expression using antibody and antibody plus blocking peptide. The figure demonstrates that the addition of blocking peptide greatly diminishes the detection of SK1 by IHC. D: Example of S1P1 expression using antibody and antibody plus blocking peptide. The figure demonstrates that the addition of blocking peptide greatly diminishes the detection of S1P1 by IHC.
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2010 American Society for Investigative Pathology Terms and Conditions

4. Figure 3
A: Western blots for HA-tag and myc-tag using lysates derived from HEK 293 cells transfected with myc-tagged SK1 or S1P1 or with HA-tagged S1P2 or S1P3 plasmid constructs. The results show the successful expression of epitope-tagged SK1, S1P1, S1P2, and S1P3. In addition, cell pellets were produced from HEK 293 cells that were either not transfected or transfected with myc-tagged SK1 or S1P1 or with HA-tagged S1P2 or S1P3 plasmid constructs. Cell pellets were formalin fixed, paraffin embedded and processed for IHC using anti-SK1, -S1P1, S1P2-, or S1P3 antibodies, as indicated. The figure demonstrates the specificity of the antibodies under the conditions used. B: Cell pellets demonstrating that treatment with siRNA S1P3 reduced S1P3 expression as assessed by IHC versus scrambled siRNA-treated cells.
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2010 American Society for Investigative Pathology Terms and Conditions

5. Figure 4
A: High nuclear SK1 expression is associated with shorter time to recurrence on tamoxifen (P = 0.016). B: High nuclear SK1 expression is associated with shorter disease-specific survival (P = 0.044).
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2010 American Society for Investigative Pathology Terms and Conditions

6. Figure 5
A: Example of membrane (M), cytoplasmic (C), and nuclear (N) S1P1 expression from tumors within the cohort. Magnification ×400. B: High membrane S1P1 expression is associated with shorter time to recurrence on tamoxifen (P = 0.008). C: High cytoplasmic S1P1 expression is associated with shorter disease-specific survival (P = 0.036).
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2010 American Society for Investigative Pathology Terms and Conditions

7. Figure 6
A: Example of cytoplasmic (C) and nuclear (N) S1P3 expression from tumors within the cohort. Magnification ×400. B: High cytoplasmic S1P3 expression is associated with shorter disease-specific survival (P = 0.019).
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2010 American Society for Investigative Pathology Terms and Conditions