Fig. 5: YBX3 regulates cellular ferroptosis by lysosomal degradation of SLC7A11.
A Co-immunoprecipitation assays showing interaction between HA-YBX3 and Myc-SLC7A11 in HEK-293T cells. Cell lysates were immunoprecipitated with anti-HA or anti-Myc antibodies and analyzed by western blotting using indicated antibodies. B Confocal images showing the co-localization of HA-YBX3 and Myc-SLC7A11 in HEK-293T cells. HA-YBX3 is labeled in red, Myc-SLC7A11 is labeled in green, and the merged image shows their overlap. Scale bar: 5 µm. C Transiently transfect 0–2 μg HA-YBX3 plasmid and 0.5 μg Myc-SLC7A11 plasmid into HEK-293T cells, collect cells 48 hours later for protein expression analysis. D CHX chase assay assessing Myc-SLC7A11 protein stability in the presence or absence of HA-YBX3 overexpression in HEK-293T cells. Protein levels were measured at indicated time points and quantified (right panel). E HEK-293T cells transfected with HA-YBX3 were treated with proteasome inhibitor MG132 or lysosomal inhibitor CQ, followed by western blot analysis of SLC7A11 expression. F Set up two experimental groups: one group will be transfected with ECFP-YBX3 and EGFP-SLC7A11, while the other group will not be transfected with ECFP-YBX3. After 48 hours of culture, stain the lysosomes using the Lyso TrackerTM probe and analyze the samples using confocal microscopy. Nucleus were stained with Hoechst. Scale bar, 5 μm. G Detect the protein expression level of SLC7A11 after transient overexpression of YBX3 and transient knockdown of ATG5 in cells. H Western blot analysis of SLC7A11 and YBX3 levels in HCT116 cells with stable USP5 knockout and shYBX3. I In USP5 overexpressing cells, transiently overexpress YBX3 and detect the protein level of SLC7A11. J Cell viability measured by CCK-8 assay in sgCtrl, sgUSP5 and sgUSP5+shYBX3 HCT116 cells, treated with DMSO, Erastin (15 μM), or Erastin + Fer-1 (5 μM) for 48 h (n = 3). K Using BODIPY-C11 staining and flow cytometry to assess lipid peroxidation (lipid ROS) levels in three treatment groups; right panel shows quantification of mean fluorescence intensity (n = 3). L Cell viability measured by CCK-8 assay in EV, Flag-USP5 and F lag-USP5 + HA-YBX3 HCT116 cells, treated with DMSO, Erastin (20 μM), or Erastin+Fer-1 (5 μM) for 48 h (n = 3). M Using BODIPY-C11 staining and flow cytometry to assess lipid peroxidation (lipid ROS) levels in three treatment groups; right panel shows quantification of mean fluorescence intensity (n = 3). Data are shown as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA.
