Fig 1: Contribution of GOT1 and GOT2 to cellular sulfite production. A. Western Blot of HEK293 lysates confirming knockout of SO, GOT1 and GOT2 (n = 3). B. Sulfite standard curve used for the determination of sulfite concentration from cellular extracts (n = 3). C. Sulfite measurement from cell extracts of WT and SUOX-/- cells with and without addition of purified SO (n = 4). Sulfite levels were normalized to cellular protein concentration. Error bars indicate standard deviation. Two-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. D. Sulfite measurement from cell extracts of WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells (n = 4). Sulfite levels were normalized to cellular protein concentration. Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. E. Relative (isoform-unspecific) GOT activity determined from extracts of WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells (n = 3). Values were adjusted to cellular protein concentration and normalized to WT. Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. pvalue: *** <0.001; ** <0.01; * <0.05; ns > 0.05. F. Relative cell proliferation of WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells determined by MTT assay (n = 3). Values were adjusted to cellular protein concentration and normalized to WT. Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.
Fig 2: Expression levels of proteins involved in H2S metabolism. A. Expression levels of proteins involved in cysteine catabolism (SO, SQR, CBS, CSE, TST, GOT1, MPST, GOT2 and ETHE1) in WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells. Tubulin was used as a loading control. B. Quantification of expression changes of SQR in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.C. Quantification of expression changes of ETHE1 in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. D. Quantification of expression changes of CSE in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. E. Quantification of expression changes of CBS in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. F. Quantification of expression changes of GOT1 in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. G. Quantification of expression changes of GOT2 in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. H. Quantification of expression changes of MPST (cytosolic and mitochondrial isoforms) in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.
Fig 3: Determination of cellular levels of H2S, persulfidated cysteine and –glutathione in SUOX-/-cells. A. Putative production of persulfidated cysteine (CysSH) and –glutathione (GSSH). B. Determination of H2S levels from WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells. Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. C. Determination of total free cysteine, free cysteine persulfide and the percentage of persulfidated cysteine from WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells. Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. D. Determination of total glutathione, glutathione persulfide and the percentage of persulfidated glutathione from WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells. Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.
Fig 4: Dependence of sulfite production on cellular cysteine- and CDO levels. A. Sulfite measurement from cell extracts of WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells after incubation in different cysteine concentrations (n = 4). Sulfite levels were adjusted to cellular protein concentration. Values were normalized to WT. Error bars indicate standard deviation. Two-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. B. Sulfite measurement from cell extracts of WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells following overexpression of CDO (n = 4). Sulfite levels were adjusted to cellular protein concentration. Values were normalized to WT. Error bars indicate standard deviation. Two-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.
Supplier Page from Abcam for Anti-Sulfite oxidase antibody [EPR7618]