Supporting Information

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Gene Array Analysis. Total RNA was isolated from cells, reverse transcribed, amplified with biotinylated nucleotides, fragmented, and hybridized against the Affymetrix U133A oligonucleotide arrays. The arrays were analyzed using MAS 5.0 (Affymetrix) with the default parameters for the statistical algorithm and probe set scaling (set to 500). The data were then filtered so that the absolute value of the fold change was >2.0 and the change P value was <0.001. Genes that were scored as absent in experimental and baseline files as well as genes scored as increasing but absent in the experimental file were removed. Using an in-house program, the list of up- and down-regulated genes from each cell line were compared and only those genes that were either increased or decreased, respectively, were included.

Fig. 6. (A) Electrophoretic mobility-shift assay (EMSA) (± anti-Flag supershift) of nuclear extracts from pBabe-Stat3-C infected HMLHT population (pB-3C) and from single-cell subclones with low (L) and high (H) Stat3- C expression. (B) Soft agar assay with HMLHT cell lines described in A, and pBabe control-infected HMLHT cells (pB) (mean ± SD).

Fig. 7. (A) Matrix metalloproteinase-9 (MMP-9) protein expression in cell culture medium from pBabe (pB)- and pBabe-Stat3-C (pB-3C)-infected MCF-10A and HMLHT cells shown by anti-MMP-9 immunoprecipitation/Western blot. (B) Gelatin zymography from cell culture medium derived from HMLHT cells expressing pBabe (pB), high levels of Stat3-C (3C4 and 3C24), low levels of Stat3-C (3C5), and pBabe MMP9 (MMP9) as a positive control. An anti-Flag Western blot of extracts from the same cell lines showing differing levels of Stat3-C expression is shown below the zymography.

Fig. 8. An alamarBlue proliferation assay of Stat3-C-expressing HMLHT cells grown in monolayer in the presence of DMSO (D) or increasing concentrations in mM of the MMP-2/9 inhibitor. The assay was performed in triplicate (mean ± SD).