Thermo scientific phusion flash high fidelity pcr master mix

For construction of HEGBC overexpression plasmid, HEGBC full-length sequences were PCR amplified using Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix (Thermo-Fisher Scientific, Waltham, MA, USA) and subcloned into the BamH I and EcoR I sites of the pcDNA3.1 plasmid (Invitrogen), termed as pcDNA3.1-HEGBC. The sequences of the primers were as follows: 5’-CGGGATCCGGGAAATGAGGACCACC-3' (forward) and 5’-GGAATTCAATATGCAAAACTTTACATTTTAGTG-3' (reverse). The empty plasmid pcDNA3.1 was used as negative control. Two pairs of cDNA oligonucleotides repressing HEGBC expression were designed, synthesized, and inserted into the SuperSilencing shRNA expression plasmid pGPU6/Neo (GenePharma, Shanghai, China), termed as shHEGBC-1 and shHEGBC-2. The target sites are 5’-GGAGCTTCCAGAAGTGGTTTC-3' and 5’-GCTGATGAGAGACATGTTTGT-3'. A scrambled shRNA was used as negative control and termed as shControl.
For construction of HEGBC stably overexpressed GBC cells, pcDNA3.1-HEGBC or pcDNA3.1 was transfected into SGC-996 and NOZ cells, and then the cells were selected with 800 μg/mL neomycin for 4 weeks. For construction of HEGBC stably depleted GBC cells, shHEGBC-1, shHEGBC-2, or shControl was transfected into GBC-SD and EH-GB2 cells, and then the cells were selected with 1 μg/mL puromycin for 4 weeks.

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.

The cDNA encoding the CDS of NFATC3 and full length of linc00423 was PCR-amplified by Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix (Thermo) and subcloned into the EcoRI and XhoI sites, BamHI and EcoRV sites of the pcDNA3.0 vector (Invitrogen) respectively, named NFATC3 and linc00423. The siRNAs specifically targeting linc00423 and NFACT3, and control siRNA were synthesized by GenePharma (Shanghai). Cells were transfected with the plasmids or siRNAs using Lipofectamine 2000/3000 (Invitrogen) according to the manufacturer’s protocol.