Fig 2: KSRP mediates the protective effects on inflammation activation, insulin resistance and atherosclerosis in FBXW2 deficiency mice. FBXW2fl/flLysmCre-/- or FBXW2fl/flLysmCre+/- mice received an injection of lentivirus encoding shRNA against KSRP and then were fed with HFD for 12 weeks (n = 15). Fasted mice were then subjected to the A) GTT and B) ITT. C) Acute insulin signaling in epididymal fat (epiWAT), the soleus, and the liver were detected by immunoblot analysis of AKT phosphorylation. D) Representative images of CD68 immunohistochemistry in epiWAT sections. Scale bars, 50 µm. The relative quantification of the adipocyte area and CD68 level are shown. Relative mRNA levels of inflammatory factors (IL-1ß, iNOS, TNFa, CCL2 and IL-6) in E) epiWAT and F) the liver of mice. G) The plasma concentrations of factors (IL-1ß, CCL2, TNFa and IL-6) in the mice were tested. ApoE-/-FBXW2fl/flLysmCre-/- and ApoE-/-FBXW2fl/flLysmCre+/- mice received an injection of lentivirus encoding shRNA against KSRP and then were fed a WD for 10 weeks (n = 15). H) Representative CD68+ immunofluorescence staining and quantitation in the plaques of aortic root. Scale bars, 200 µm. I) Relative mRNA levels of inflammatory mediators (TNFa, IL-1ß, iNOS, CCL2 and IL-6) in the aortas of mice. The data are analyzed by ANOVA with the post-hoc test and are presented as the mean ± SEM. *p < 0.05 and # p < 0.05.

Fig 3: FBXW2 (P3) attenuates KSRP degradation and inflammation in vitro. A) Immunoprecipitation followed by western blot was performed to detect the KSRP ubiquitination in HEK293T cells cotransfected with Flag-KSRP and Myc-FBXW2 plus HA-K48-Ub constructs (FL, ?C, ?N, and ?F). The ?F plasmid lacks the F-box domain. ?C and ?N plasmid indicate separate depletion of the C-terminal and N-terminal WD40 repeat regions. B) Schematic diagram of the interaction between KSRP and the SCFFbxw2 complex. C) Immunoblot analysis of the V5-FBXW2 protein after immunoprecipitation of KSRP with anti-Flag antibody. HEK293T cells were cotransfected with Flag-KSRP and V5-FBXW2 in different combinations with Myc-FBXW2 constructs (P1, 1–138 aa; P2, 139–306 aa; P3, 307–454 aa of FBXW2). D) Immunoprecipitation followed by western blot was performed to detect KSRP ubiquitination. HEK293T cells were transfected with different combinations of the indicated plasmids. E) The relative mRNA expression of proinflammatory cytokines (IL-1ß, TNFa, iNOS, CCL2, and IL-6) was measured after LPS stimulation for 12 h in the absence or presence of the indicated segments. The data are analyzed by ANOVA with the post-hoc test and are presented as the mean ± SEM. *p < 0.05 and # p < 0.05.

Fig 4: FBXW2 mediates the ubiquitination and degradation of KSRP. A) KSRP expression in FBXW2fl/flLysmCre‐/− and FBXW2fl/fl LysmCre+/‐ peritoneal macrophages was tested by western blot assay (n = 5). B) Representative immunofluorescence images of KSRP and CD68 in epiWAT sections from FBXW2fl/flLysmCre‐/− and FBXW2fl/flLysmCre+/− mice on HFD for 12 weeks and the relative quantification (n = 10). Scale bars, 20 µm. C) Immunoblot analysis of the KSRP level in the lysates of FBXW2fl/flLysmCre‐/− and FBXW2fl/flLysmCre+/‐ peritoneal macrophages treated with cycloheximide (CHX; 20 µg mL−1) for the indicated hours (n = 5). D) KSRP was coexpressed with empty vector or different contents of FBXW2 plasmids in Raw264.7 cells with or without MG132 treatment. The expression levels of KSRP and FBXW2 were analyzed using western blotting (n = 5). E) Real‐time qPCR assay analysis of KSRP in FBXW2fl/flLysmCre‐/− and FBXW2fl/flLysmCre+/‐ peritoneal macrophages (n = 5). F) HEK293T cells were transfected with Myc‐FBXW2 or Myc‐ΔF‐box‐FBXW2 plus vectors for HA‐Ub and Flag‐KSRP and then were treated with MG132. The cell lysates were subjected to immunoprecipitation with Flag M2 beads and western blotting with anti‐HA antibody (n = 5). G) Raw264.7 cells were transduced with Scr shRNA or FBXW2 shRNA plus vectors for HA‐Ub and Flag‐KSRP and then were treated with MG132. The protein extracts were subjected to immunoprecipitation with Flag M2 beads and western blotting with anti‐HA antibody (n = 5). H) Sequence alignment of KSRP with the phospho‐degron sequences recognized by FBXW2. I) HEK293T cells cotransfected with Myc‐FBXW2 plus vectors for Flag‐KSRP (WT) or Flag‐KSRP (3A). Western blot analysis for KSRP expression in the cell lysates (n = 5). J) HEK293T cells were pretransfected with Myc‐FBXW2 plus Flag‐KSRP (WT), Flag‐KSRP (3A) or HA‐Ub, and then treated with MG132. Immunoprecipitation analysis for KSRP ubiquitination with anti‐Flag‐M2 beads (n = 5). K) Myc‐FBXW2 plus Flag‐KSRP(WT) or Flag‐KSRP (3A) were first transfected into HEK29T cells. Then cells were treated with CHX (20 µg mL−1) for the indicated hours. Immunoblot analysis of the KSRP levels in the cell lysates (n = 5). L) FBXW2 promotes KSRP ubiquitination via K48 linkage. Immunoblot analysis of His‐tag pull‐down and cell extracts derived from HEK293T cells transfected with the indicated constructs. The data are analyzed by Student's t test or ANOVA with the post‐hoc test and are presented as the mean ± SEM. *p < 0.05.

Fig 5: FBXW2 (P3) mitigates inflammation activation, insulin resistance and atherosclerosis in vivo. After 4 weeks of HFD feeding, the C57BL/6J mice were injected with adeno-associated virus carrying HA-tagged human FBXW2 (P3) to mediate FBXW2 (P3) expression in macrophages and then were fed an HFD for another 8 weeks (n = 15). Fasted mice were subjected to the A) GTT and B) ITT. C) Acute insulin signaling in epididymal fat (epiWAT), the soleus, and the liver were detected by immunoblot analysis of AKT phosphorylation. D) Representative images of CD68 immunohistochemistry in epiWAT sections. Scale bars, 50 µm. The relative quantification of the CD68 level is shown. Relative mRNA levels of inflammatory mediators (IL-1ß, TNFa, iNOS, CCL2 and IL-6) in epididymal fat (epiWAT) (E) and epiWAT F40/80+ macrophages (F) of mice. G) Plasma concentrations of inflammatory factors (TNFa, IL-1ß, CCL2, and IL-6) in the mice were tested. After 4 weeks of WD feeding, the ApoE-/- mice were injected with adeno-associated virus carrying HA-tagged human FBXW2 (P3) to mediate FBXW2 (P3) expression and then were fed a WD for another 6 weeks (n = 15). H) Whole aortas were stained by Oil Red O staining. I) Histological analysis and J) representative CD68+ immunofluorescence staining of the lesions in aortic root sections, and the relative quantitation (n = 8). Scale bars, 200 µm. K) Relative mRNA levels of inflammatory factors (TNFa, IL-1ß, iNOS, CCL2, and IL-6) in the aortas of mice. L) Plasma concentrations of inflammatory mediators (TNFa, IL-1ß, CCL2, and IL-6) in the mice were tested. The data are analyzed by ANOVA with the post-hoc test and are presented as the mean ± SEM. *p < 0.05.