Journal: Communications Biology
Article Title: Lactate orchestrates the TGFβ pathway and ferroptosis nexus in organ fibrosis via USP2 lactylation
doi: 10.1038/s42003-025-09286-z
Figure Lengend Snippet: A Total cell lysates from WT TECs subjected to Co-IP with anti-SIRT2 or anti-USP2 antibody, and western blotting using indicated antibodies (left). HK2 cells were transfected with Ad HA- SIRT2 and/or Ad-Flag USP2 as indicated. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies (right). B WT TECs transfected with Ad-null or Ad- Sirt2 as indicated, followed by TGF-β1 stimulation. Total cell lysates were subjected to Co-IP with anti-USP2 antibody, and western blotting using the indicated antibodies. C HK2 cells transfected with Ad-Flag Usp2 WT, K139R, K205R, K241R, K340R, K420R, K447R, K464R, or K473R as indicated. The total cell lysates were prepared and western blotting using indicated antibodies. D The conservation of USP2 K447 in different species. E WT TECs transfected with Ad-Flag Usp2 WT and then incubated with TGFβ1 for the indicated time. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. F Total cells lysates from the kidney of UUO or FA-treated mice subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. G WT TECs transfected with Ad-Flag Usp2 WT or Ad-Flag Usp2 K447R in the presence or absence of TGFβ1. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. H – L WT TECs transfected with Ad-null, Ad- Usp2 WT, and Ad- Usp2 K447R under TGFβ1 stimulation was indicated. Representative images of SMAD3 immunofluorescence staining are shown in ( H ; (Scale bar = 25 μm)), and densitometry quantification of nuclear levels of SMAD2 are shown in ( I ). J Western blot analyses of SMAD3 in the fractions extracted from TECs. K 3TP-Lux luciferase activity assay in HEK293T cells after transfection of the 3TP-Lux plasmid, a renilla plasmid, and Ad-null, Ad- USP2, or Ad- USP2 K447R for 24 h, followed by the treatment with or without 2 ng/ml TGFβ1 for 16 h. Relative luciferase activity is presented as folds of that in the cells with transfection of Ad-null ( n = 5). L Western blot analysis of TGFBR1, USP2, SMAD3, pSMAD3, and Tubulin in TECs (left). The mRNA level of Col1a1 , Col1a2 , Serpine1 , and Smad7 as determined by qPCR (right; n = 5). M WT TECs transfected with Ad-Flag- Usp2 and Ad- Sirt2 or si Sirt2 in the presence or absence of TGFβ1 as indicated. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. N WT TECs transfected with Ad HA- Sirt2 or Ad-Flag- Usp2 under TGFβ1 stimulation as indicated. Total cell lysates were subjected to Co-IP with anti-HA antibody, and western blotting using the indicated antibodies. O WT TECs transfected with Ad-Flag- Usp2 WT, Ad-Flag- Usp2 K447R, or Ad- Sirt2 under TGFβ1 stimulation as indicated. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. P HK2 cells transfected with Ad-Flag- USP2 WT, Ad-Flag- SIRT2 H187Y, or Ad HA- SIRT2 under TGFβ1 stimulation as indicated (Elements in this figure were created with BioRender.com). Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. Q USP2-SIRT2 docking with the HDOCK server. High magnification of the boxed areas is presented on the left. The arrow indicates K447 of the USP2 protein. R Total cells lysates from the kidney of UUO and FA-treated WT or S2KO mice subjected to Co-IP with anti-USP2 antibody, and western blotting using the indicated antibodies. For all panels, data were presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by a one-way ANOVA with a Bonferroni correction test.
Article Snippet: Mice were divided into groups ( n = 6 per group) receiving AAV9-empty vector (AAV9-Ctrl), AAV9- Ksp - Usp2 ( Usp2 OE group), AAV9- Ksp - Usp2 K447R (mutant group), and AAV9- Ksp -short hairpin RNA targeting Usp2 (AAV-shUsp2); all AAV constructs were provided by GeneChem (Shanghai, China).
Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Incubation, Immunofluorescence, Staining, Luciferase, Activity Assay, Plasmid Preparation