Fig 2: IRF5 promoted the binding of transcription factors SP1, PU.1 to glycolytic gene promoters. (A) The binding of SP1 to glycolytic gene promoters HK1, TPI1, PGAM1, PFKFB3 and positive controls IL‐6, IFN‐α after siRNA‐IRF5 intrathecally in CCI mice by ChIP; (B) the binding of PU.1 to glycolytic gene promoters and positive controls after siRNA‐IRF5 intrathecally in CCI mice by ChIP; (C) the binding of SP1 to glycolytic gene promoters and positive controls after siRNA‐IRF5 administration in cultured primary microglia by ChIP; (D) the binding of PU.1 to glycolytic gene promoters and positive controls after siRNA‐IRF5 administration in cultured primary microglia by ChIP.

Fig 3: Knockout of STX4 in brown adipocytes leads to age-dependent involution of brown adipose tissue and activation of pyroptosis.a The representative image of different fat pad from 5.5 - month - old WT and Ucp1-STX4KO male mice. EPI, epididymal white adipose tissue; BAT, interscapular brown adipose tissue; SUB, subcutaneous white adipose tissue. b Brown adipose tissue mass in WT and Ucp1-STX4KO male mice at 2 months (2M), 5 months (5M), and 9 months (9M) of age. For WT mice, n = 6 (2M), n = 13 (5M), n = 4 (9M). For Ucp1-STX4KO mice, n = 6 (2M), n = 9 (5M), n = 4 (9M). p = 0.0036 (2M), p = 2.01E-08 (5M), p = 6.1E-05 (9M). c Core body temperature of WT and Ucp1-STX4KO male mice (2 months of age) maintained at room temperature and then shifted to 4 °C for 60 min. n = 8 (WT), n = 5 (Ucp1-STX4KO) mice. p = 0.006 (0 min), p = 0.0004 (10 min), p = 0.0003 (20 min), p = 8.6E-05 (30 min), p = 0.0001 (40 min), p = 0.0002 (50 min), p = 0.0014 (60 min). d The expression of pyroptotic genes in brown adipose tissue of Ucp1-STX4KO and WT male mice by RNAseq. n = 4 mice. e Brown adipose tissue from 2- month - old WT and Ucp1-STX4KO male mice was extracted and subjected to RT-qPCR to determine the indicated mRNA levels. n = 6 mice. p = 1.68E-06 (Casp1), p = 0.03 (Casp11), p = 0.005 (Nlrp1b), p = 0.725 (Nlrp3), p = 0.007 (Aim2), p = 0.004 (Il18), p = 0.004 (Adgre1), p = 0.011 (Tnfa). f The activity of cleaved Caspase1 in BAT of Ucp1-STX4KO and WT male mice by immunostaining of cleaved Caspase1 antibody. g Quantitated signal of cleaved Caspase1 by HistoQuant. n = 5 (WT), n = 4 (Ucp1-STX4KO) mice. p = 0.024. h, i Representative immunoblotting of brown adipose tissue from 2- month - old WT and Ucp1-STX4KO male mice with different antibodies. PGAM1 as loading control. j Perilipin immunofluorescence (red) and DAPI (blue) staining of nuclei in brown adipose tissue of WT and Ucp1-STX4KO male mice at 2 months of age. Arrows indicate perilipin-depleted cells. Scale bars: 100 µM. k F4/80 immunostaining in brown adipose tissue in WT and Ucp1-STX4KO male mice at 2 months of age. Scale bars: 200 µM. All data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by two-tailed Student’s t test.

Fig 4: Comparative proteomic analysis of whole mouse livers and iHLCs(A) Venn diagram depicting overlap of significant DE proteins in APOE4 vs. APOE3 across whole mouse livers and iHLCs (batch 1 and 2).(B) Gene Ontology Biological Process (GO-BP) and KEGG pathway analysis of the 70 proteins shared across mouse liver and iHLCs, showing protein counts per category.(C) Heatmap of proteins consistently upregulated or downregulated in APOE4 vs. APOE3 across whole mouse liver and iHLCs (batch 1 and 2).(D–E) Western blot analysis of CYP27A1, FDFT1, and PGAM1 in APOE4 and APOE3 whole mouse livers, with relative densitometry quantification. n = 16 per group.(F–G) Western blot analysis of CYP27A1, FDFT1, and PGAM1 in APOE4 and APOE3 isogenic iHLCs, with relative densitometry quantification. n = 6 per group.(E and G) Statistical significance was determined by unpaired t test. Data are shown as mean ± SD. ∗p < 0.05.

Fig 5: NFS1 regulates glycolytic pathway in AAA progression. MOVAS were stimulated with 3 µM Ang II for 24 h. NaHS (30 µM) was added 30 min prior to Ang II treatment where applicable. A) PFKP, HK2, ENO1, PGAM1, LDHA, ALDOA and PKM2 protein expressions assessed by Western blotting in MOVAS. B–H) Quantification of protein expressions measured by Western blotting in MOVAS (n = 6). All data was analyzed by one‐way ANOVA followed by the Tukey post hoc test. I) Differences in extracellular lactate contents between various treatments (n = 6). Data were analyzed by one‐way ANOVA followed by the Tukey post hoc test (extracellular lactate). J) Mitochondrial superoxide content in different groups measured by MitoSOXs staining, the results were showed as fluorescence images. Scale bar, 50 µm. K) Representative fluorescence images of mitochondrial permeability transition pore evaluated by mPTP staining. Scale bar, 50 µm. L) JC‐1 staining tested by microplate reader with different treatments (n = 6). Data were analyzed by one‐way ANOVA followed by the Tukey post hoc test.