Fig. 5

SIRT1 is degraded by hypoxia-induced autophagy through the p62-SIRT1 autolysosome pathway. a RT-qPCR analysis of SIRT1 mRNA in HMrSV5 cells exposed to hypoxic conditions for 0 h, 6 h, 24 h. b Immunoblot analysis of p62, LC3I/II and SIRT1 at the indicated time points during hypoxia. c Immunofluorescence analysis of SIRT1 localization in hypoxic PMCs after 0 h, 6 h, or 24 h. Scale bar represents 10 μm. d The nuclear-cytosol distribution experiment detected the nuclear and cytoplasmic location of SIRT1 following hypoxia treatment (H), compared to normoxic cells (N). e and f Analysis of p62, LC3I/II and SIRT1 protein levels during hypoxia and in response to treatment with chloroquine (CQ) in HMrSV5 cells or ATG7 knockout in HEK293 cells. g HMrSV5 cells were cultured under hypoxic conditions for 0 h, 6 h, or 24 h. Immunoprecipitation was performed using SIRT1 or p62 antibodies, followed by immunoblotting with antibodies against SIRT1 and p62. h Immunofluorescence confocal microscopy was performed to assess the colocalization of SIRT1 and p62 at different time points during hypoxia. Scale bar represents 10 μm. i and j HEK293 cells were co-transfected with plasmids for Flag-p62 and HA-SIRT1 N terminal domain (NTD, aa 1–234), sirtuin (catalytic) domain (SD, aa 234–510), C terminal domain (CTD, aa 510–747) or full length (FL, aa 1–747). Lysates were immunoprecipitated with anti-Flag antibody