Fig. 9

eCIRP blocks TRIM32-mediated polyubiquitination and proteasomal degradation of ZBP1. a Co-IP assays demonstrate the interaction between ZBP1 and CIRP in cells treated with LPS alone or LPS with rmCIRP for 12 h. The presence of CIRP and ZBP1 in the precipitated complexes is confirmed by Western blotting. b Proximity ligation assay (PLA) reveals physical associations between ZBP1 and CIRP as red spots in WT primary MPVECs after 12 h of co-treatment with LPS alone or LPS and rmCIRP (scale bar = 20 μm). The bar chart shows the quantitative analysis of PLA signals from panel B shows a significant increase in ZBP1-CIRP association when co-treated with LPS and rmCIRP compared to LPS alone. c Immunofluorescence images display the localization of CIRP (red) and ZBP1 (green) in cells treated with LPS, with and without rmCIRP treatment. The merged and enlarged images highlight the colocalization of CIRP and ZBP1, which is enhanced with rmCIRP treatment (scale bar = 20 μm). The right images show the fluorescence intensity profiles for CIRP and ZBP1 along the lines marked “i” and “ii” that support the colocalization observation. The bar graph quantifies the colocalization coefficients, showing a significant increase with LPS and rmCIRP treatment. d Western blotting analysis monitors the stability of ZBP1 protein over time in the presence of LPS and LPS + rmCIRP, indicating that rmCIRP treatment enhances ZBP1 stability compared to LPS treatment alone. A line graph quantifying the relative ZBP1 levels suggests that rmCIRP preserves ZBP1 stability against LPS-induced degradation. The symbol *** indicates statistically significant differences compared to the control group. ^***P < 0.001. e Western blotting analysis to monitor the stability of ZBP1 protein following CHX inhibition of protein synthesis in the presence of LPS, LPS + rmCIRP, and MG132. f Ubiquitination assays for ZBP1, conducted with and without rmCIRP treatment in the presence of HA-tagged ubiquitin (HA-Ub), reveal a pattern of polyubiquitination. The extent of ZBP1 ubiquitination is determined by immunoblotting using antibodies specific for HA-tagged polyubiquitin conjugates. The expression levels of TRIM32, CIRP, and ZBP1 are confirmed by analyzing 5% of the input from cell lysates. The bar chart below displays the quantification of polyubiquitinated ZBP1 levels, normalized to the control. g PLA detects physical associations between ZBP1 and TRIM32 as red spots in MPVECs after 12 h of co-treatment with LPS and rmCIRP or LPS alone (scale bar = 20 μm). The right image shows the quantitative analysis of PLA signals revealing a significant decrease in ZBP1-TRIM32 association with LPS and rmCIRP co-treatment compared to LPS alone. h Immunofluorescence images show the localization of TRIM32 (red) and ZBP1 (green) in cells treated with LPS, with and without rmCIRP treatment for 12 h. The merged and enlarged images illustrate the colocalization of TRIM32 and ZBP1 (scale bar = 20 μm). Fluorescence intensity profiles for TRIM32 and ZBP1 along the lines marked “i” and “ii” in the right mages support the colocalization data. The bar graph quantifies the colocalization coefficients, showing a significant reduction with LPS and rmCIRP treatment compared to LPS treatment alone. The data are presented as the mean ± SD. ^**P < 0.01, ^***P < 0.001. eCIRP extracellular cold-inducible RNA-binding protein, TRIM32 tripartite motif-containing 32, ZBP1 Z-DNA binding protein 1, LPS lipopolysaccharide, rmCIRP recombinant mouse cold-inducible RNA-binding protein, PLA proximity ligation assay, WT wild type, MPVEC mouse pulmonary vascular endothelial cell, CHX cycloheximide, MG132 proteasome inhibitor, HA-tag hemagglutinin-tag, HA-Ub hemagglutinin-tagged ubiquitin, Co-IP co-immunoprecipitation