Fig. 9: Identification of potential GPSM1 inhibitors.
From: GPSM1 impairs metabolic homeostasis by controlling a pro-inflammatory pathway in macrophages
a SPR assay with Biacore diagram and saturation curve of AN-465/42243987 binding to GPSM1 protein. As AN-465/42243987 concentration increases, the chip RU continues to increases. b–f BMDMs treated with 50 μM AN-465/42243987 or vehicle control for 16 h and then stimulated with LPS or not. b Representative immunofluorescence images indicating P65 nuclei translocation showed by high-content screen. BMDMs were stained for P65 (red) and DAPI (blue). Scale bars, 100 μm. c Quantification of the proportion of P65 nuclear translocation exhibited in (b) (n = 3 independent samples per condition). d The mRNA abundance of A20 in BMDMs after LPS stimulation for 20 min (n = 4 independent samples per condition). e The mRNA abundance of pro-inflammatory markers in BMDMs stimulated with LPS for 3 h (n = 3 independent samples per condition). f The effect of AN-465/42243987 on P65 translocation in GPSM1f/f and GPSM1f/f; Lyz2-cre BMDMs (n = 3 or 4 independent samples per condition). Positive% was defined as the index (P65 intensity in nuclei/cytoplasm) > 1.5. g–n In vivo effects of AN-465/42243987. DIO mice (already HFD-fed for 8 weeks) were peritoneally administered with either 0.5 mg/kg AN-465/42243987 or vehicle, twice per week for 4 weeks. g Body weights (n = 10 biologically independent mice per group). h Tissue weights (n = 10 biologically independent mice per group). i GTT and AOC (n = 7 biologically independent mice per group). j ITT and AOC (n = 7 biologically independent mice per group). k Oil Red O staining. Scale bars, 100 μm. l H&E and F4/80+ IHC staining of eWAT. Scale bars, 100 μm. m Representative flow cytometry analysis and quantification of the expression of CD11c from CD45+F4/80+ cells in SVFs from eWAT (n = 4 biologically independent mice per group) and scWAT (n = 3 biologically independent mice per group). n RT-PCR analysis indicating mRNA abundance in eWAT (n = 9 biologically independent mice per group). Independent experiments were performed three times with similar results (b, k, l). All data are presented as means ± SEM. P values are determined by unpaired two-tailed Student’s t-test (d, e, h to j, m, and n), or the two-way ANOVA with Sidak’s multiple-comparisons test (g, i, and j) or one-way ANOVA with Tukey’s multiple-comparisons test (c).
