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. 2022 Jan 31;12(2):236. doi: 10.3390/biom12020236

Figure 2.

Figure 2

Effect of leptin on SPX expression via STAT3 activation. To determine the effect of leptin on SPX expression, eight-week-old male mice were fasted for 24 h and icv-injected with leptin (2.5 μg); tissue samples were collected 1 h after leptin injection. (A,B) Total RNA was isolated from the mediobasal hypothalamus (MBH) (A) and arcuate nucleus (ARC) (B) and analyzed with qRT-PCR. (C) Leptin was icv-injected into ObRb-Cre;Rpl22HA mice to analyze leptin-induced SPX mRNA level in ObRb-expressing cells. SPX mRNA was determined using qRT-PCR of MBH RNA samples immunoprecipitated with HA antibody. (D) Mice were icv-injected with 100 μM of S3I-201, an inhibitor of STAT3 activation, and then after 3 h, icv-injected with leptin. After 1 h, MBH SPX mRNA was analyzed by qRT-PCR. (E) ChIP assays were performed to verify whether STAT3 directly binds to the SPX promoter and whether leptin affects this binding. Mice were icv-injected with either saline (CTL) or leptin (LEP) and nuclear DNA samples from their MBH were immunoprecipitated with anti-STAT3 antibody or mouse IgG. Immunoprecipitates were analyzed by PCR amplification using a primer set specific to the mouse SPX promoter. Input represents the DNA extracted from the mouse MBH before immunoprecipitation. Data are presented as mean ± S.E.M. *, p < 0.05; ***, p < 0.001.