Fig. 7: CO-induced Beclin 1 protects against neuronal cell death by preventing necroptosis via suppressing MLKL oligomerization.
From: CO confers neuroprotection via activating the PERK-calcineurin pathway and inhibiting necroptosis
a, b SH-SY5Y cells were pretreated with 1 μM GSK2606414 for 30 min and then treated with 20 μM CORM-A1 for 12 h. Beclin 1 protein expression (a) and Beclin 1 mRNA expression (b) were determined by western blotting and qRT-PCR, respectively. c Beclin 1 expression was detected by western blotting in SH-SY5Y cells treated with 20 μM CORM-A1 after the cells were transfected with CA-GSK-3β or pretreated with 1 μM GSK2606414 (left). Quantification of Beclin 1 is shown in the right panel. d SH-SY5Y cells were transfected with scRNA or siTfeb and then treated with 1 mM MPP+ and 20 μM CORM-A1 for 24 h. The levels of TFEB and Beclin 1 protein expression were analyzed by western blotting. e SH-SY5Y cells were treated with 1 mM MPP+ for 24 h after pretreatment with 20 μM CORM-A1 for 1 h. Reducing samples were subjected to western blotting for detection of p-MLKL, MLKL, and Beclin 1. To confirm MLKL oligomerization, analysis of non-reducing samples was performed by western blotting using an MLKL antibody. f SH-SY5Y cells were pretreated with 20 μM CORM-A1 for 2 h, followed by treatment with 1 mM MPP+ for 24 h. Cells were immunostained with anti-p-MLKL and anti-Beclin 1 antibodies, and counterstained with DAPI. g SH-SY5Y cells were transfected with scRNA or siBECN1 and then treated with 1 mM MPP+ for 24 h in the presence or absence of 20 μM CORM-A1. Reducing samples were subjected to western blotting for detection of p-MLKL, MLKL, and Beclin 1. To confirm MLKL oligomerization, analysis of non-reducing samples was performed by western blotting using an MLKL antibody. h Mice (n = 5) were subjected to MPTP (25 mg/kg) injection in the presence or absence of CO inhalation (250 ppm) for 13 days. Starting on day 4, mice were treated with MPTP once daily for 7 consecutive days. TH and p-MLKL expression in SNc were determined by immunofluorescence, scale bar: 10 μm. Data were expressed as mean ± SD; *p < 0.05 and ***p < 0.001.
