Vitronectin Destroyed Intestinal Epithelial Cell Differentiation through Activation of PDE4-Mediated Ferroptosis in Inflammatory Bowel Disease

Figure 2

VTN promoted ferroptosis to suppress cell differentiation. (a) Intestinal epithelial cells were digested and reseeded in a 6-well plate overnight; after 12-serum-free treatment, the cells were treated with or without VTN (5 μg/ml) for another 48 hr, real-time PCR were performed to analyze GPX4 and SLC7A11 expression at mRNA level, data presented as the mean ± s.e.m. of three independent experiments and were analyzed by t test, . (b) CaCO₂ and HT-29 cells were treated and described as (a). The total protein was collected and subjected by western blotting to detect GPX4 and SLC7A11 expression at protein level; the band intensity was measured and quantified by t test, data presented as the mean ± s.e.m. of three independent experiments and were analyzed by t test, , . (c) CaCO₂ cells were treated described as (a), and the ROS level was detected according to the manufacturer’s instruction. (d) After starvation, CaCO₂ and HT-29 cells were treated as indicated for 48 hr, and the total protein level was collected; WB was employed to detected activation of ferroptosis by RSL3 on intestinal epithelial cell differentiation. (e) After serum starvation for 24 hr, CaCO₂ and HT-29 cells were treated with or without VTN, followed by ferroptosis inhibitor ferrostatin-1 (Fer) (10 μM) stimulation for further 48 hr. The total lysates were collected to analyze indicated protein levels. (f) 24 hr after transfection with GPX4 plasmid, CaCO₂ cells were treated with or without VTN for another 24 hr to collect the total protein; WB was performed to detect indicated proteins. (g) CaCO₂ cells were treated as (f), and ROS staining was used to analyze the ROS level.

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