Fig 2: (A) NS1 fluorescence (excitation at 465 nm, emission at 620 nm) in selected organs of the mice after sacrifice at 4 h and 24 h post-IP relative to vehicle alone and their quantification. (B) NOX2 levels at 4 h in these organs determined by Western blots and their quantification relative to amino black presented in Supplementary Figure S7. The staining with amido black of the total proteins transferred onto the nitrocellulose membrane is used to normalize the levels of the protein of interest (i.e., NOX2) in each tissue analyzed. This normalization was used because any housekeeping proteins, such as ß-actin or ß-tubulin, show variable levels between tissues. Statistical significance indicated by stars (** p-value < 0.01) was evaluated by a two-way ANOVA followed by Tukey’s multiple comparisons test. A significant accumulation of NS1 was observed in the kidney after 4 h post-injection while at 24 h post-injection, NS1 fluorescence was significantly increased in the pancreas. No significant fluorescence of NS1 was noted in all other tissues analyzed.

Fig 3: Mechanism dissection on how ERß regulates NOX2 expression. (a) NOX2 mRNA levels were regulated by E2 or ERß antagonists in HK-2 and HKC-8 cells. Cells were treated for 6 hr with vehicle (control), 0.75 mM oxalate, 0.75 mM oxalate + 10 nM E2, 0.75 mM oxalate + 10 nM E2 + 10 µM ICI, or 0.75 mM oxalate + 10 nM E2 + 10 µM PHTPP for q-PCR of NOX2 mRNA levels. Bonferroni's test was applied for statistical analysis of results. (b) Predicted 3 potential EREs located in the 3 kb-NOX2 promoter region. (c) ChIP was performed in HK-2 cells, and only ERE I of the NOX2 promoter could be bound by ERß. (d) ERE I wild-type or mutant of NOX2 promoter reporter constructs were cotransfected with pRL-TK at a ratio of 1000 : 1 into HK-2-shLuc and HK-2-shERß cells. Luciferase reporter assay was performed after 48 hr incubation. For (a) and (d), data shown are mean ± SD. * P < 0.05 and ** P < 0.01. n.s. = not significant.

Fig 4: Comparison of the binding of NS1-2 (A, shown in cyan) and NS1-3 (B, shown in yellow) to a model of NOX2. For clarity, only FAD, and residues involved in the ligands binding are shown. ADE stands for adenine. (C) Zoom-in of the phosphate interaction with lysine/arginine residues, driving the recognition of NOS and NOX.

Fig 5: Effect of NS1 and its isomers on (A) eNOS activity determined by the hemoglobin assay [19]; (B) Representative absorption changes around 340 nm due NOX2-induced NADPH consumption using NOX2 = 2.5 µM reconstituted with 50 µM FAD, NADPH = 50 µM with or without 25 µM of NS1-2 or NS1-3 in 20 mM Tris buffer pH = 7.5, 150 mM NaCl.