Fig 1: Contribution of ASM to the autophagic degradation of GPX4. hFob1.19 cells were treated with HG (35 mM) in the presence or absence of ASM-shRNA, HCQ (10 µM) and NAC (5 mM). Following the treatment, A Western blot analysis of GPX4 expression was carried out. Relative density of each protein bands were quantified, and the data are presented as the mean ± SD of three independent experiments. ****P < 0.0001. B Atg5 protein levels inhFob1.19 cells demonstrated by Western blotting. C hFob1.19 cells were treated with HG (35 mM) in the presence or absence of Atg5-shRNA. Western blot analysis of GPX4 was carried out. D Beclin 1 protein levels in hFob1.19 cells verified by Western blot analysis. E hFob1.19 cells were treated with HG (35 mM) in the presence or absence of Beclin 1-shRNA. Western blot analysis of GPX4 was carried out. F ASM-shRNA-transfected hFob1.19 cells were treated with HCQ (10 µM) for 12 h. Western blot analysis of GPX4 was carried out. All data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001
Fig 2: Effect and potential mechanism of ASM on T2DOP. Sixty SD rats were divided into four groups (n = 15). A Determination of iron ion levels in serum. B MDA measurement in serum. C Determination of ASM activity in serum. D Detection of ceramide in serum. E IHC for ASM, Beclin 1, LC3 and GPX4 in four groups of rats. Scale bar = 100 μm. ****P < 0.0001
Fig 3: ASM is involved in HG-induced ferroptosis. ASM-shRNA-transfected hFob1.19 cells were treated with NG (17.5 mM) or HG (35 mM) for 48 h. hFob1.19 cells were treated with HG in the presence or absence of Fer-1 (5 µM). Following the treatment, A ROS generation was demonstrated by the DHE assay kits. B LPO was detected using C11 BODIPY 581/591 fluorescent probe. Scale bar = 20 μm. C LPO by MDA assay was determined. D LPO was detected by the 4-HNE assay kits. All data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig 4: Autophagy level and ASM/ceramide system expression were measured in hFob1.19 cells. Cells were treated with NG (17.5 mM) and HG (26.25 mM, 35 mM, 43.75 mM, respectively) for 48 h. A hFob1.19 cells expressing ptfLC3 were treated with different conditions, and fluorescent images were captured using LSCM. The number of autophagosomes represented by yellow puncta and autolysosomes represented by red puncta in merged images. Scale bar = 20 μm. B Western blot analysis of indicated proteins were analyzed. C Western blot analysis of LC3 II was carried out. D Ceramide levels were measured in hFob1.19 cells. All data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig 5: Pivotal role of ASM in the regulation of HG-induced autophagy for the execution of ferroptosis. hFob1.19 cells were treated with HG (35 mM) in the presence or absence of ASM-shRNA, HCQ (10 µM) and NAC (5 mM). Following the treatment, A ASM expression was determined using Western blot analysis. B Ceramide levels were measured. C Western blot analysis of LC3 II was carried out. D Western blot analysis of indicated proteins were carried out. E Autophagic flux was determined by ptfLC3 fluorescent probe. Scale bar = 20 μm. F LPO by C11 BODIPY 581/591 fluorescent probe was assessed. Scale bar = 20 μm. G ROS generation was detected by DHE assay kits. Scale bar = 20 μm. H LPO was determined by MDA assay. I LPO was detected by the 4-HNE assay kits. All data are presented as the mean ± SD of three independent experiments.****P < 0.0001
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