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. 2021 Mar 30;65(2):214–221. doi: 10.1165/rcmb.2020-0374OC

Figure 3.

Figure 3.

Blockade of STAT3 signaling decreased leptin induction of Trpm7 gene transcription, promoter activity, and promoter demethylation through alteration of the pSTAT3 binding in PC12LEPRb cells. (A) PC12LEPRb cells were treated with 1 ng/ml leptin and/or 5 μM SD1008 for 3 days. The Trpm7 mRNA level and promoter methylation were assessed by using qPCR and bisulfite sequencing, respectively. To measure promoter activity, PC12LEPRb cells were transfected with pEZX-Luc-Trpm7 or pEZX-Luc CTL vector, which was followed by treatment with 5 μM SD1008, 1 ng/ml leptin, or combined treatment with SD + Leptin for 3 days. Luc activity was examined. Data are represented as a ratio of the RLU of the pEZX-Luc-Trpm7 to the RLU of the CTL vector pEZX-Luc. Binding of (B) PolIIA (RNA polymerase IIA) and (C) pSTAT3 at R1, R2, and R3 clusters in the Trpm7 promoter were assayed by using ChIP-qPCR. PC12LEPRb cells were treated with 1 ng/ml leptin and/or 5 μM SD1008 for 3 days before the ChIP assay. Results are shown as the percent change in binding as compared with the PC12LEPRb CTLs. All data are present as mean ± SEM values. A one-way ANOVA with Tukey multiple comparisons was used to determine the significant differences between groups. **P < 0.01, ***P < 0.001, and ****P < 0.0001. SD + Leptin = combined treatment with SD1008 and leptin.