Overexpression inhibits sccoc cell growth. 1x10

Figure S1: (A) Overexpression inhibits SCCOC cell growth. 1x10⁶ Tu167 or JMAR cells were infected with 5 moi of S100A7 or control GFP adenovirus for 48 h in 6 well plates, and then passed into 10 cm dishes and were cultured for another 48 h before harvested for cell counting. (B) Western blotting of SCCOC cell lines under sub-confluent culture conditions using antibodies as indicated. MDA-MB-468 cells serves as a positive control. Upper panel, 80 g protein extracts; middle and bottom panels, 20 g protein extracts.

Supplementary results: -Catenin is an integral component of E-Cadherin-mediated adherens junctions, playing important roles in both cell adhesion and Wnt/-Catenin signaling (Nelson & Nusse, 2004). Interestingly, we found that transient transfection of S100A7 siRNA into MDA-MB-468 decreased S100A7 expression and also reduced the cell membrane assembly of E-Cadherin as shown by immunofluorescent staining (Fig. S2A), suggesting that downregulation of S100A7 not only increased -Catenin signaling, but also inhibited E-Cadherin-mediated cell adhesion. Consistent with this idea, S100A7 shRNA stable clones exhibited a more scattered morphology and did not adhere to one another as well as control transfected clones (Fig. S2B). Western blotting further confirmed that in S100A7 shRNA cells, the level of E-Cadherin expression was lower than that in the control shRNA cells (Fig. S2C). To further explore the possible mechanisms of E-Cadherin suppression by down-regulation of S100A7, we examined the expression of Snail, which represses the expression of E-Cadherin (Cano et al., 2000), in the stable clones. Consistent with these observations, Snail expression was found to be higher in S100A7 shRNA stable polyclones than in control shRNA transfected polyclonal cells (Fig. S2C), which might account for the decreased expression of E-Cadherin in S100A7 shRNA stable clones. Finally, we also examined the effect of decreased expression of S100A7 in JMAR cells. Similar to MDA-MB468 cells, membrane assembly of E-Cadherin was reduced by S100A7 siRNA transfection as shown by immunofluorescent staining (Fig. S2D). Western blotting of the Triton X-100 insoluble cell membrane fraction also indicated that cell membrane-associated E-Cadherin was decreased by S100A7 siRNA transfection (Fig. S2E). Taken together, these results indicated not only that downregulation of S100A7 enhanced -Catenin signaling, but also that S100A7 was required for the efficient assembly of E-Cadherin to the cell membrane, and down-regulation of S100A7 impaired E-Cadherin-mediated cell-cell adhesion.


Figure S2. Decreased expression of S100A7 leads to increased levels of Snail, and decreased E-Cadherin-mediated cell adhesions. (A) Representative confocal microscopic image of immunoflorescent staining of S100A7 (green) and E-Cadherin (red) in MDA-MB468 cells 48 h after transient siRNAs transfection. (B) Representative microscopic image of MDA-MB 468 lentiviral shRNA stable clones #2 and control clones #5 under 10% serum cultured conditions. (C) Western blotting of MDA-MB-468 lentiviral shRNA stable cells with different antibodies as indicated. (D) Representative confocal microscopic image of immunoflorescent staining of E-Cadherin (red) in JMAR cells 48 h after transient siRNAs transfection. (E) Western blotting of JMAR 48 h after transient siRNAs transfection with different antibodies as indicated. The Triton X-100-insoluble fraction (see Materials and Methods) was isolated for measuring cell membrane-associated E-Cadherin.


Figure S3. Representative IHC images of tumors derived from JMAR control (A) and S100A7 (B) shRNA stable polyclones. In each panel, consecutive tumor sections stained with different antibodies are shown as indicated, in which 100X images on right hand side magnified box areas of the 40X images on left hand side. Note that S100A7-expressing area (*) has a higher level of E-Cadherin, but reduced levels of -Catenin and PCNA staining when compared with adjacent S100A7-negative areas.

References

Cano A, Perez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, et al. (2000). The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2: 76-83.

Nelson WJ,Nusse R. (2004). Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303: 1483-7.