Pgl3 basic vector

Mouse IL-2 promoter region (747 bp) was cloned into pGL3 Basic vector by GenScript. R2D^fl/fl or Lck^CreR2D^fl/fl CD4⁺ T cells were nucleofected with IL-2 promoter by using Amaxa Nucleofector electroporation, rested overnight, and stimulated with plate-bound anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) for 24 hours. Then, cells were harvested and lysed, and luciferase activity was detected by GloMax-Multi Detection System (Promega) using the Dual-Luciferase Reporter Assay Kit (E1910, Promega). Luciferase activity was normalized to renilla luciferase activity, as well as to the R2D^fl/fl CD4⁺ T cells.

The promoter of HEGBC containing the predicted p-STAT3 binding sites was PCR amplified using Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix (Thermo-Fisher Scientific) and subcloned into the Kpn I and Xho I sites of the pGL3-basic vector (Promega), termed as pGL3-HEGBC-pro. The sequences of the primers were as follows: 5’-GGGGTACCCTATTGCTGCACTCACACACCC-3′ (forward) and 5’-CCGCTCGAGCGCCAGAGCCCAAGCTATC-3′ (reverse). The empty vector pGL3-basic was used as negative control. The p-STAT3 binding sites mutated HEGBC promoter was synthesized by GenScript (Nanjing, China) and subcloned into the Kpn I and Xho I sites of the pGL3-basic vector, termed as pGL3-HEGBC-pro-mut. The constructed luciferase reporter plasmids were cotransfected with the pRL-TK plasmid expressing renilla luciferase into NOZ cells. 12 h later after transfection, the NOZ cells were treated with 5 μM SC144 or 20 ng/mL IL-11 for 72 h. Then the luciferase activity was measured using Dual-Luciferase® Reporter Assay System (Promega) in accordance with the manufacturer’s instruction.