Fig. 5: Fascin interacts with xCT and decreases its stability.

A Induction of ferroptosis-related proteins upon Fascin knockdown and overexpression in MDAMB231 cells. Cell lysates were collected after 72 h of transfection. B Western blot analysis showed that Fascin knockdown increased xCT levels, which was reversed by re-expression of Fascin. C Western blot analysis of xCT protein levels in MDAMB231 cells treated with NP-G2-044 (5, or 10, 15 μM) for the indicated times (24, 48, or 72 h). xCT mRNA was detected in Fascin knockdown (D) or Fascin-overexpressing (E) cells by quantitative qRT-PCR analysis. F Western blot analysis of xCT levels in MDAMB231 cells treated with CHX (100 μg/ml) with or without MG132 (10 μM) and CQ (25 μM) for 16 h. G, H Fascin knockdown or overexpression cells were treated with 100 μg/ml CHX for the indicated times. Relative xCT protein levels were quantified with ImageJ. I, J MDAMB231 cell lysates were subjected to immunoprecipitation (IP) with antibodies towards Fascin or xCT or with control immunoglobulin G (IgG). Immunoblot assays were performed using Fascin and xCT antibodies. K Immunoprecipitation of cell lysates with Fascin knockdown or incubated with NP-G2-044 (5 μM) cells was performed with xCT antibody, after which lysates were immunoblotted with antibodies against Ub, xCT, Fascin, and β-actin. L Immunoblot analysis of Fascin, xCT, and β-actin in MDAMB231 cells transfected with control or Fascin siRNAs with or without xCT siRNAs for 72 h. M MDAMB231 cells transfected with control or Fascin siRNAs with or without xCT siRNAs were incubated with the indicated concentrations of erastin for 48 h and then assayed for cell viability. n.s. not significant. *p < 0.05, **p < 0.01, and ***p < 0.001. The data are displayed as the means ± s.d of three independent experiments.