Stat3 c flag prc cmv

CT26.WT (ATCC CRL-2638TM), HT-29 (ATCC HTB-38) and HCT 116 (ATCC CCL-247) cell lines were grown till 70% confluence in DMEM medium supplemented with 10% FBS, 1% sodium pyruvate and 1% penicillin/streptomycin (all from Gibco-Thermo Fisher Scientific, Waltham, USA) at 37 °C in humidified incubator containing 5% CO₂. H₂O₂ (Sigma Aldrich) working solution was prepared just before adding. Cells were treated with 200 μM H₂O₂ for 24 h; when indicated, pharmacological inhibitors (Additional file 3: Table S3) were added to the cell culture 1 h prior to H₂O₂ treatment. CT26.WT were transfected with Stat3-C Flag pRc/CMV or pRc/CMV (Invitrogen-Thermo Fisher Scientific) as control. Stat3-C Flag pRc/CMV was a gift from Jim Darnell (Addgene plasmid # 8722). The transfection was performed on 60% confluent cells using Lipofectamine2000 transfection agent (Invitrogen-Thermo Fisher Scientific) according to the manufacturer instructions.

Constitutively active STAT3 construct STAT3-C Flag pRc/CMV and STAT3 reporter construct 4xM67 pTATA TK-Lu were purchased from Addgene (United States). Transfection of STAT3C plasmids into HepG2 cells was performed as described previously (Su et al., 2017 (link)). Cells were transfected with plasmids for 48 h before functional assays were carried out. For luciferase reporter assay, cells were co-transfected with STAT3 reporter construct 4xM67 pTATA TK-Lu plus Renilla luciferase expression vector PRL-CMV (Promega, United States) using the lipofectamine 2000 reagent (Invitrogen, United States). After 24-h transfection, cells were treated with vehicle or EEAC (60, 90, 120 μg/ml) for another 24 h. Firefly and renilla luciferase activities were detected using the dual-luciferase reporter assay system (Promega, United States). Firefly luciferase activity was normalized to renilla luciferase activity in cell lysate as described elsewhere (Tse et al., 2017 (link)).