Supplemental Data. Supplemental Data contains three figures. Supplemental data Figure 1 shows an example of the HPLC technique used to measure BH4:BH2 throughout the manuscript. Supplemental data Figure 2 shows a time course of changes in BH4:BH2 in MCF7 cells after addition of SP. This established the time course used in the in vitro experiments. Supplemental data Figure 3 compares the effects of two NOS inhibitors on SP-increased cGMP in MCF-7 and MDA-231 cells. Supplemental data Figure 4 shows the response of MDA-231 xenografts to >30 days treatment with SP in terms of tumor growth measured as tumor volumes and by a Kaplan Meier analysis of animal survival.

Acidic Alkaline pterin biopterin Supplemental Figure 1. Colonic BH4/BH2 measurements by acidic alkaline oxidation (Fukushima T and Nixon JC (1980) Chromatographic analysis of pteridines. Methods Enzymol 66: ). Top panels are HPLC chromatograms from control colons while the bottom panels are chromatograms from control colons treated with peroxynitrite. Under acidic conditions BH4 and BH2 are converted to biopterin while under alkaline conditions BH2 remains biopterin while BH4 is converted to pterin. The ratio of BH4:BH2 changes from 9.1:1 to 1.7:1 after treatment with peroxynitrite.

Supplemental Figure 2. Time Course for SP-induced changes in BH4:BH2 in MCF-7 cells. The BH4:BH2 of cultured MCF-7 cells was measured by the HPLC technique shown in Supplemental Figure 1 at various after adding 20µM SP at t = 0.. Results are reported as the mean ± SEM. n=3. These results demonstrate that MCF-7 cells achieve the highest BH4:BH2 within 6 hrs of SP treatment and that elevated BH4:BH2 is sustained for at least 16 hrs.

Supplemental Figure 3. Relative efficacy of L-NNA and the iNOS selective inhibitor 1400W on cGMP formation in MCF-7 and MDA231 cells. cGMP is measured after incubating for 6 hrs with 20µM SP ± 200nM L-NNA, SP ± 200 nM 1400W or SP ± both inhibitors. Data reported as the mean ± SEM, * p<.001, n=3, for SP relative to control and & p <0.01, n=3, relative to SP alone. Positive controls are cells treated with the NO donor, GSNO, at 100 μM for 2 hrs. * &

Supplemental Figure 4. SP significantly reduces the rate of MDA-231 tumor xenograft growth and enhances animal survival. A. Xenografts were created in athymic nu/nu mice by subcutaneous injection of 10 6 MDA231 cells into the hind flanks. Once tumors were established animals were divided into 2 groups as indicated. SP was provided from day 0 at 0.64mg/kg/day in drinking water. Tumor volume is calculated according to the formula: tumor volume (mm 3 ) = (L x W 2 )/2, where L is the larger dimension and W is the smaller dimension in millimeters from microcaliper measurements. Data presented as mean relative volume ± SEM. Number control animals =7; 11 tumors, SP=6 animals; 10 tumors. *=p<0.05, ***=p<0.01. B. Survival of MDA231 xenograft bearing mice. Kaplan-Meyer survival plot of animals receiving either 0.64mg/kg/day SP or not. Animals receiving SP survived significantly longer than control animals determined by log-rank analysis (p<.05, n=13, 21 tumors). A. B. * ***