Fig 1: IL-32 protein half-life is regulated by the oxygen sensor ADO. (A) JJN-3 cells were transfected with ADO- and nontargeting Ctrl siRNA. After 24 h, the cells were seeded and cultured overnight in normoxia or hypoxia before being treated with 5 μg/ml CHX and the IL-32 CHX chase assay in normoxia and hypoxia. One representative WB of IL-32 and ADO siRNA-treated cells of n = 5 independent experiments is shown. (B) JJN-3 cells were transfected with ADO and nontargeting Ctrl siRNA. After being transfected for 24 h, the cells were cultured overnight in hypoxia before being treated with 5 µg/ml CHX and reoxygenized in normoxic culture conditions. Cells were harvested at indicated time points.
Fig 2: Time course of induction of known hypoxia-inducible proteins. Five cell lines; SH-SY5Y (A), RKO (B), HepG2 (C), Kelly (D), and EA.hy926 (E) were exposed to 1% O2 for the indicated periods of time. Representative immunoblots (top) and mRNA levels of the ADO substrates (RGS5, RGS4, and IL-32) are shown below. All data represent the mean ± SD from three independent experiments.
Fig 3: Interplay between transcriptional and proteolytic regulation of IL-32.A, induction of IL32 mRNA in HepG2 cells treated for 16 h with TNFα (20 ng/μl). B, Accumulation of IL-32 protein in HepG2 cells treated with TNFα prior to exposure to hypoxia for the times indicated. C, ADO-competent or -deficient HepG2 cells were treated with TNFα or IL-1β (20 ng/μl) for 16 h then exposed to hypoxia for a further 2 h, and levels of IL-32 protein were analyzed. HIF-1α protein levels were assayed for comparison. All data represent the mean ± SD from three independent experiments, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, Mann–Whitney t test (A) or 2-way ANOVA with Holm-Sidak post hoc analysis (B and C).
Fig 4: Coordinated regulation of RGS4 by HIF-2α and ADO under hypoxia.A, SH-SY5Y cells were transfected with siRNA targeting HIF-1α and/or HIF-2α, or scrambled control (scr), and subjected to 24 h of hypoxia (1% O2). RGS4 mRNA levels were assessed alongside canonical HIF-1α (CA9) or HIF-2α (VEGF) target genes as positive controls. The color scale represents mean fold change relative to cells treated with scr control (normoxia) from three independent experiments. B, ADO-competent or -deficient SH-SY5Y cells were exposed to hypoxia (1% O2) or treated with the PHD inhibitor FG-2216 (100 μM), or both, for 4 h and samples blotted for RGS4 and HIF-1α protein. RGS4 mRNA levels were assessed in parallel, confirming transcript upregulation. Immunoblots are representative of three separate experiments, and data in the histogram are the mean ± SD, n = 3. All treatments significantly induced RGS4 mRNA, 2-way ANOVA with Holm-Sidak post-hoc analysis, p < 0.001.
Fig 5: Lack of apparent feedback regulation in the ADO pathway during hypoxic exposure.A, levels of ADO, arginyl transferase 1 (ATE1), and HIF prolyl-hydroxylase 3 (PHD3) protein in SH-SY5Y exposed to hypoxia for 4, 24, or 48 h. B, hypoxic upregulation of mRNA transcript encoding PHD3, but not ADO, in SH-SY5Y cells. C, no effect of hypoxia was observed on the transcript levels of other components of the Arg/Cys N-degron pathway (METAP1, methionine aminotransferase 1, UBR1/2, Ubiquitin Protein Ligase E3 Component N-Recognin 1/2). All data represent the mean ± SD from three independent experiments.
Supplier Page from Abcam for Anti-ADO antibody [EPR6581]