Short interfering rnas sirnas

Phosphate-buffered saline (PBS), Roswell Park Memorial Institute medium (RPMI) 1640, and fetal bovine serum (FBS) purchased from Hyclone (Logan, UT, USA) were used. Rabbit polyclonal antibodies against human HERG1 and TXNDC5 were purchased from Abcam (Cambridge, UK), whereas those against human p21, cyclin D1, E-cadherin, vimentin, fibronectin, Janus N-terminal kinase (JNK)1/2, phosphorylated (p)-JNK1/2, p38, p-p38, phosphoinositide 3-kinase (PI3K), p-PI3K, AKT, p-AKT, Src, p-Src, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, used as the internal control) were purchased from Cell Signaling Technology (Danvers, MA, USA). Short interfering RNAs (siRNAs) that targeted TXNDC5 were obtained from GenePharma (Shanghai, China). Invitrogen (Carlsbad, CA, USA) was the supplier of TRIzol, Moloney murine leukemia virus reverse transcriptase (M-MLV-RT), and Lipofectamine 2000, whereas Roche (Penzberg, Germany) provided SYBR Green I Master kits. Unless otherwise mentioned, biochemical reagents used were from Sigma (St. Louis, MO, USA).

Short-interfering RNAs (siRNAs) targeting MTX1 or CISD1 were synthesized by Shanghai Genepharma Co., Ltd, Shanghai, China. The sequences were as follows: siNC: 5′-UUCUCCGAACGUGUCACGU-3′; siMTX1: 5'-CCCACAUUCUCAGUCUCUA-3'; siCISD1-1: 5'-CAGCUGCAAUUGGUUAUCU-3'; siCISD1-2: 5'-CGUGAAGUUACCUGAUUGU-3'.
For gene overexpression, FLAG-tagged MTX1 plasmid was constructed through cloning MTX1 cDNA (GeneBank Accession Number: NM_002455.5) into pcDNA3.1 vector. CISD1 overexpression plasmid (also FLAG tagged) was constructed based on pENTER vector, which was obtained from Vigene Biosciences, Shandong, China. MTX1-overexpressing lentivirus was prepared by Shanghai Genepharma Co., Ltd, Shanghai, China.