Fig 1: Gene and protein expression levels in THP1 cells treated for twenty-four hours. mRNA (A) and protein (B) expression levels of UCN1, UCN2, UCN3, CRH, and Spexin were respectively measured by quantitative real-time PCR and Western blots in THP1 cells treated with high glucose (25 mM), palmitate (200 µM) and the combination for 24 h (n = 3 for each condition). Data are normalized to internal GAPDH and presented as fold-change compared with control data. One-way ANOVA with repeated measurements followed by Sidak´s post hoc test were used to determine the significance of the differences in means between the groups (* p < 0.05, ** p < 0.01, *** p < 0.001).
Fig 2: CRH increases Gal3 expression in vitro. (A): CRH increases Gal3 levels in BV2 cells (* p < 0.05); (B): CRH increases Gal3 levels in PM (* p < 0.05); (C,D): CRH increases the numbers of Gal3 puncta in Mg and increase the percentage of Gal3 puncta positive in Mg (* p < 0.05, scale bar = 10 µ). For Western blots, all experiments were independently repeated at least three times for statistical analysis.
Fig 3: CRHR2 blockage mitigates CRH-mediated effects on Mg. (A): CRH has no effects on CRHR2 levels in BV2 cells; (B): As-2B mitigates CRH-mediated upregulation ofGal3 (* p < 0.05 vs. control, # p < 0.05, vs. CRH treatment); (C): As-2B mitigates CRH-mediated upregulation effects on CD11n (* p < 0.05 vs. control, # p < 0.05, vs. CRH treatment); (D): As-2B mitigates CRH-mediated upregulation effects on mCat B (* p < 0.05 vs. control, # p < 0.05, vs. CRH treatment). All experiments were independently repeated at least three times for statistical analysis.
Fig 4: Gene expression levels in PBMCs according to body weight. mRNA expression levels of UCN1, UCN2, UCN3, CRH, and spexin were measured by quantitative real-time PCR in PBMCs from normal weight (n = 8), overweight (n = 10), and obese children (n = 22). Data are normalized to internal GAPDH and presented as fold-changes compared with normal weight data. One-way ANOVA followed by Bonferroni´s post hoc test were used to determine the significance of difference in means between groups (* p < 0.05, ** p < 0.01). NW (•), OW (¦), and OB (?).
Fig 5: Gene expression in THP1 cells treated for four hours. mRNA expression levels of UCN1, UCN2, UCN3, CRH, and spexin were measured by quantitative real-time PCR in THP1 cells treated with high glucose (25 mM), palmitate (200 µM) or the combination for 4 h (n = 3 for each condition). Data are normalized to internal GAPDH and presented as fold-change compared with control data. One-way ANOVA with repeated measurements followed by Sidak´s post hoc test were used to determine the significance of the differences in means between the groups (* p < 0.05, ** p < 0.01, *** p < 0.001).
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