Cocl2

GAS (SMB00313), CoCl₂ (232696), and calcium ionophore (A23187) were purchased from Sigma-Aldrich (St Louis, MO, USA). GAS and CoCl₂ were dissolved in phosphate-buffered solution to prepare the original solution of 100 mM, which was stored at -20° C. CQ diphosphate (HY-17589) and 3-BDO (HY-U00434) were purchased from MedChemExpress. Fluo-4AM (S1060) was obtained from Beyotime Biotechnology, Inc. (Nanjing, China). Primary antibodies against β-actin (66009-1-Ig, 1:10 000), LC3 (14600-1-AP, 1:1000), p62 (18420-1-AP, 1:1000), p62(66184-1-lg), LAMP-2 (66301-1-lg, 1:1000), and CaMKIIα (11533-1-AP, 66843-1-Ig) were purchased from Proteintech (Wuhan, China). Rabbit anti-p-p62 (Thr349) (Ab211324) was purchased from Abcam (Cambridge, MA, USA). Primary antibodies against CaMKIIα (#4436, 1:1000) and anti-p-CaMKIIα (Thr286) (#12716, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti-rabbit IgG (H&L)-HRP (BS13278, 1:10000) and goat anti-mouse IgG (H&L)-HRP (BS12478) were obtained from Bioworld. Goat PAb to Rb IgG Alexa Fluor®488 (Ab150077, 1:200) and goat PAb to MS IgG Alexa Fluor®647 (Ab150115) were purchased from Abcam. A fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (A0516, 1:200) was purchased from Beyotime Biotechnology.

MCF-7 cells were transiently transfected with the indicated plasmids. Twenty-four hours later, the cells were treated with 10 µM MG132 (Selleckchem, Houston, USA) and 150 μM CoCl₂ (Sigma-Aldrich, St. Louis, MO, USA) prior to cell lysis for indicated time (0, 4, 8, 12, 16 h). To test the half-life of HIF1α, the transfected MCF-7 cells were firstly treated with 150 μM CoCl₂ for 24 h, then the cells were incubated with 50 μg/ml cycloheximide (CHX; MedChemExpress, USA), a kind of protein synthesis inhibitor, for the indicated periods of time (0, 1, 3, 6 h). Finally, the above whole-cell lysate was subjected to western blot analysis.

The differentiated PC12 cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). PC12 cells were maintained in dulbecco’s modified eagle medium (DMEM, Gibco, United States) containing 10% FBS (Gibco, United States) at 37°C in a humidified incubator with 5% CO₂.
Cellular hypoxia was induced by CoCl₂ (C8661, Sigma-Aldrich, St. Louis, MO, United States), which is a chemical compound widely used to induce hypoxic-ischemic condition by increasing the generation of ROS (Wang et al., 2000 (link)). In vitro, PC12 cells were treated with CoCl₂ for 24 h to induce hypoxia. Myricetin was dissolved in DMSO with a 20 mM stock concentration. According to the preliminary concentration gradient experiments, 200 μM myricetin was selected as the optimal concentration to treat the cells. The myricetin group was pretreated with myricetin 2 h before CoCl₂ stimulation, while the ML385-treated group was pretreated with NRF2 inhibitor ML385 (HY-100523, MedChemExpress, Monmouth Junction, NJ, United States) (20 μM) for the same time. After the pretreatment, CoCl₂ was added to each group with myricetin or ML385 according to the experimental requirements.