Fig. 6: WWP2 regulates IRF7 transcriptional activity in macrophages through non-degradative ubiquitination.

a Representative WB of co-immunoprecipitation experiment with WWP2 (upper) or IRF7 (lower) showing a direct interaction of WWP2 with IRF7 in BMDMs following LPS stimulation. WCL, whole cell lysate. b Expression of WWP2 (green) and IRF7 (red) in BMDMs following LPS stimulation. The intensity profiles of WWP2 and IRF7 were plotted along an ideal straight line (yellow) crossing the nuclei of two representative cells, showing the WWP2 and IRF7 signals overlap (bottom right). c In-cell ubiquitylation analysis of IRF7 in BMDMs from control WT and WWP2^−/− mice. Cells were treated with MG132 (10uM, 3 hrs) followed by LPS stimulation. d Representative WB measuring p-IRF7 protein expression in WT and WWP2^−/− BMDMs with or without LPS stimulation. Quantification of p-IRF7 protein expression is shown in the bar plot below. e Quantification of the IRF7 Interferon-stimulated response element (ISRE) luciferase activity in WT and WWP2^−/− BMDMs following LPS treatment. n = 7–11 for each group. f Representative immunoblots of native Polyacrylamide gel electrophoresis (PAGE) identifying monomeric and dimeric forms of IRF7 (indicated by arrows) in WT and WWP2^−/− BMDMs with or without LPS stimulation. g Representative cellular images (BMDMs) of in-cell IRF7 and p-IRF7 from 10,000 acquired events by imaging flow cytometry (see Methods), showing typical externalized and internalized patterns of colocalized or separately distributed IRF7 and p-IRF7. h, i Imaging flow cytometry shows IRF7 (h) and p-IRF7 (i) DRAQ5 similarity in WT and WWP2^−/− BMDMs following LPS treatment, using a similarity score cutoff ≥1.5 for protein nuclear translocation (left panels). Quantification of nuclear translocation of IRF7 and p-IRF7 (right panels). n = 4 for each group. j Representative WB showing IRF7 and p-IRF7 protein distribution in nuclear fractions of BMDMs (with or without LPS) from WT and WWP2^−/− mice. k Schematic of the proposed mechanism through which WWP2 regulates IRF7 and the Ccl5/Ly6c^high monocyte axis during the early phase of fibrogenesis, which in turn affects cardiac tissue fibrosis at a later stage of the fibrogenic process. In each experiment, LPS stimulation (100 ng/ml, 4 hrs). Unless otherwise indicated, data are shown as dot plots with mean ± SD, and statistical significance is assessed by the non-parametric Mann–Whitney U test.