SUPPLEMENTARY DATA
FIGURE LEGENDS FOR SUPPLEMENTAL FIGURES
Fig. S1. Quantitative RT-PCR analysis of SF and Akt-regulated gene expression (T=6 hr).
Subconfluent proliferating DU-145 cells were transfected with DN-Akt or pcDNA3 vector, washed,
allowed to recover for several hours, incubated  SF (100 ng/ml) for T=6 hr, and then harvested for
quantitative RT-PCR assays (see Methods section). For each time point, expression of N=8 different
SF-inducible genes was quantitated relative to control conditions (pcDNA3, no SF) and corrected for
expression of the control gene (-actin). The values plotted are means  SEMs of N=3-4 independent
experiments for each time point. Abbreviations: MX1, WNT4, AMPD3, MMP1, ECM1, and STC1
(see Fig. 1 legend); MMP10 [matrix metalloproteinase 10 (stromelysin 2)] and EPHB2 (ephrin
receptor B2).
Fig. S2. Quantitative RT-PCR analysis of SF and Akt-regulated gene expression (T=48 hr).
Assays were performed as described in Fig. S1 legend, except that a 48 hr incubation period with SF
was utilized rather than a 6 hr incubation. Abbreviations: See Fig. S1 legend.
Fig. S3. Quantitative RT-PCR analysis of genes induced by DN-Akt (T=6 hr). DU-145 cells were
treated as described in Fig. S1 legend and harvested for quantitative RT-PCR assays. Expression of
N=5 different DN-Akt-inducible genes was quantitated relative to control conditions (pcDNA3, no SF)
and corrected for expression of -actin. Values shown are means  SEMs of N=3-4 independent
experiments. Abbreviations: BRCA1 (breast cancer susceptibility gene-1, splice variant), IFIT1
(interferon-induced protein with tetratricopeptide repeats 1), G1P3 [interferon -inducible protein
(clone IFI-6-16), transcript variant 3], MEK5C (mitogen-activated protein kinase kinase 5, isoform C),
and PLXDC1 (plexin domain containing 1).
Fig. S4. Quantitative RT-PCR analysis of genes induced by DN-Akt (T=48 hr). Assays were
performed as described in Fig. S3 legend, except that a 48 hr incubation period with SF was utilized
rather than a 6 hr incubation. Abbreviations: See Fig. S3 legend.
Fig. S5. Knockdown of expression of four SF and c-Akt regulated genes by RNA interference.
Small interfering (si) RNAs and the corresponding control siRNAs were prepared for four genes
shown to be induced by SF in a c-Akt dependent manner: WNT4, MX1, AMPD3, and EPHB2. To
experimentally validate these siRNAs, subconfluent proliferating DU-145 cells were pre-treated with
gene-specific or control siRNAs (50 nM) for 48 hr and then treated  SF (100 ng/ml) for T=6 hr or
T=48 hr and harvested for RNA isolation and quantitative RT-PCR analysis. For each time point, the
mRNA values were expressed relative to the control gene (-actin) and normalized to the controltreated cells (+pcDNA3, 0 SF). The values plotted based one or two independent experiments at each
time point.
Fig. S6. Effects of SF, ADR, and siRNAs on gene expression under MTT assay conditions. To
determine if the expected changes in gene expression due to SF and the gene-specific siRNAs persisted
under the conditions used for MTT assays, DU-145 cells were treated as in Fig. 4 and assayed at the
end of the experiment by qRT-PCR analysis. Data are shown for AMPD3 (A), EPHB2 (B), MX1 (C),
and WNT4 (D). mRNA levels were expressed relative to -actin and normalized to the control value
(0 SF, 0 ADR, no siRNA). The data plotted are means ± ranges of two independent experiments.