Fig. 2: USP2 stabilizes and deubiquitinates TFRC to promote ferroptosis.

A The IPA Analysis of proteomics in the kidney of WT and U2KO mice. B, C WT TECs transfected with siCtrl or siUsp2 as indicated in the present with or without TGFβ1. B The total cell lysates were prepared and western blotting was performed using the indicated antibodies. C MDA, SOD, GSH, and Fe²⁺ levels in cell homogenates were detected by a commercial kit (n = 5). D, E WT TECs transfected with Ad-null or AdUsp2 as indicated in the presence or absence of TGFβ1. D The total cell lysates were prepared and western blotting using the indicated antibodies. E MDA, SOD, GSH, and Fe²⁺ levels in cell homogenates were detected by commercial kit (n = 5). F Representative images of BODIPY 581/591 C11 staining. Scale bar = 50 μm. G WT TECs transfected with AdUsp2 and siTfrc as indicated upon TGFβ1 stimulation. (Left) The total cell lysates were prepared and western blotting using the indicated antibodies. (Right) MDA, SOD, GSH, and Fe²⁺ levels in cell homogenates were detected by a commercial kit (n = 5). H WT TECs transfected with siUsp2 and AdTfrc as indicated upon TGFβ1 stimulation. (Left) The total cell lysates were prepared and western blotting using the indicated antibodies. (Right) MDA, SOD, GSH, and Fe²⁺ levels in cell homogenates were detected by a commercial kit (n = 5). I, J WT or U2KO TECs incubated with CHX (I) or MG132 (J) as indicated. The total cell lysates were prepared and western blotting using the indicated antibodies. K Co-IP assays were performed using lysates from WT TECs stimulated with TGF-β1, and western blotting using the indicated antibodies. L WT TECs transfected with Ad HA-Tfrc and/or Ad-Flag Usp2 as indicated, and then subjected to TGF-β1 stimulation. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. M HK2 cells transfected with Ad-Flag USP2 WT (Flag-WT) or Ad-Flag USP2 C276A. Total cell lysates were subjected to Co-IP with anti-TFRC antibody, and western blotting using the indicated antibodies. N The database provides TXNDC5 posttranslational modification of individual sites (left). HK2 cells transfected with shUSP2, Ad-Flag TFRC WT, K39R and K160R as indicated. The total cell lysates were prepared and western blotting using the indicated antibodies (right). O HK2 cells transfected with siRNA USP2 (siUSP2), Ad-Flag TFRC WT, or Ad-Flag TFRC K39R as indicated. The total cell lysates were prepared and western blotting using the indicated antibodies. P Schematic illustration of TFRC deletion mutants (left). The numbers indicate the amino acid positions. HA- TFRC deletion mutants were coexpressed with Flag-USP2 in HEK293T cells (right). For all panels, data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by a one-way ANOVA with a Bonferroni correction test.