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. 2023 Oct 30;68:102949. doi: 10.1016/j.redox.2023.102949

Fig. 6.

Fig. 6

Hypoxia induces per-polysulfide via AMPK-mediated phosphorylation of CSE. Panel A Conservation sequence of human CSE at S346 and T355 compared across various species, including human, monkey, Danio rerio (Zebra fish), Xenopus tropicalis (frog), rat, mouse, Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. Panel B CSE phospho sites S346 and T355 showing modular signaling domains of protein Serine/Threonine kinases motif groups using Scansite 4. Panel C Representative blots of MAEC treated under hypoxia for 0, 5, 15, 30, 60 and 90 min probed for pCSES346, total CSE, pAMPK, AMPK and GAPDH. Panel D Quantitation of pCSES346 and p-AMPK protein levels, respectively from western blots in Panel A. Panel E Representative blots of MAEC treated with mock or AMPK inhibitor (AMPK-I) Dorsomorphin under hypoxia for 30min. Panel F Quantitation of pCSES346 and pAMPK protein levels, from western blots in Panel E. Panel G MAEC treated with mock or AMPK inhibitor (AMPK-I), Dorsomorphin under hypoxia for 30 min followed by per-polysulfide (SSP4 fluorophore) signal. Panel H Representative blots from HUVEC treated under normoxia or hypoxia or hypoxia + AMPK-I for protein levels of pCSES346, total CSE, pAMPK, AMPK and GAPDH. Panel I quantitation of HUVEC blots for pCSES346 and pAMPK treated with mock or AMPK inhibitor (AMPK-I) Dorsomorphin under hypoxia for 30 min. Panel J HUVECs treated under hypoxia or hypoxia + AMPK-I for 30 min followed by per-polysulfide (SSP4 fluorophore) measurement. Densiometric analysis of p-AMPK and p-CSE346 blots were normalized to total AMPK or total CSE (normalized to GAPDH for protein). All data are averaged from triplicates from each experiment with at least n = 5. ****P < 0.0001; ***P < 0.0002; *P < 0.01.