PMC

A. Ribbon models of PHD2 showing a shared binding site for α-KG, hypotaurine and 2-HG. B. Relative docking scores for α-KG, hypotaurine, 2-HG and taurine to the shared binding site on PHD2. A more negative value indicates increased affinity for the binding site. C. Binding free energy of PHD2-mediated HIF-1α hydroxylation before and after hypotaurine binding. A more negative value indicates a more spontaneous chemical reaction.

Hypotaurine concentration in mouse placenta was measured during the course of late pregnancy (A). Data represents the mean and S.E.M of three determinations. Statistical analysis was conducted using one-way ANOVA with the Dunnett post-hoc test (control; day 12.5). Hypotaurine was continuously administered via an osmotic pump (12 mg/day/mouse) from gestational day 8.5 to pregnant Ez^+/− female mice that had been mated with Ez^+/− male mice. Plasma concentration of hypotaurine in maternal mice given hypotaurine (•) or vehicle (○) (B). Hypotaurine concentration in placenta (C) and fetus (D) of each genotype of Ez after administration of hypotaurine (hatched column) or vehicle (open column) to dams. Fetal body and placenta weights were measured at gestational day 18.5 (E, F). The number of fetuses of each genotype is shown in brackets (E). Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test. P values less than 0.05 were regarded as significant and are indicated with an asterisk (*). The distribution of hypotaurine to placenta (G) and fetus (H) was evaluated by dividing the amount shown in panel C and D by the maternal plasma concentration at gestational day 18.5 shown in panel B. Comparisons between hypotaurine (hatched column) and vehicle (open column) administered groups or between genotypes were done by using Student's t-test. P values less than 0.05 were regarded as significant and are indicated with an asterisk (*) when compared to vehicle and a cross (†) when compared to Ez^+/+.

A. Immunoprecipitation showing hydroxylated (HO-HIF-1α) forms of HIF-1α in glioma cells treated with the proteasome inhibitor MG-132, hypotaurine (0.5 or 1mM), both or neither as indicated. B. Hydroxylation assay for HIF-1α after incubation with PHD2 (EGLN1) and 0, 2.5 or 5mM hypotaurine as indicated. The y-axis indicates percentage of maximum signal. The x-axis indicates m/z ratio. Arrows indicate m/z ratio for HIF-1α; arrowheads indicate m/z ratio for OH-HIF-1α. C. Immunoprecipitation showing HIF-1/2α ubiquitination in glioma cells treated with PBS or hypotaurine as indicated. D. CHX assay of glioma cell cultures treated with PBS or hypotaurine at multiple time-points followed by Western blot for HIF-1α, HIF-2α or β-actin. E. Quantification of HIF-1/2α half-lives based on the densiometric analysis of immunoblot in (D). F. Real-time quantitative PCR of HIF targets EDN1, EPO, GLUT1 and VEGF in glioma cells incubated with 0, 0.1, 0.5 or 1mM hypotaurine as indicated.

250μM homocysteic acid resulted in a 5% decrease in cell amount A., a 48% decrease in intracellular hypotaurine B. and a 25% decrease in intracellular taurine C. Media supplemented with 200μM cysteamine results in a 12% increase in cell amount D. a 7-fold intracellular hypotaurine increase E. and unappreciable intracellular taurine change F. 45μM taurine leads to a 13% decrease in cell amount G. a 52% decrease in intracellular hypotaurine H. and a 7% increase in intracellular taurine I. Cell amount was given as mean±s.d. (n=8) and were determined by OD₄₅₀ after incubated with WST-1. Intracellular hypotaurine and taurine concentrations were given as μg/ml/(g cell dry weight) obtained through at least 3 biological and 3 technical replicates. J. Relative ADO gene expression of ADO gene silenced cell line (ΔADO) and the vector control cell line (VCT). A 45% decrease of ADO gene expression in ΔADO cells was observed (n=4). K. Compared to VCT cells, intracellular hypotaurine of ΔADO cells decreased by approximately 38% (n=6). L. Growth properties of ΔADO cells with different hypotaurine content in the media. VCT cells cultured without additional hypotaurine were used as controls. 100 μM hypotaurine in media led to growth rescue in ΔADO cells (n=8). Data was processed by two-way ANOVA.