Pcmv6 xl5

HEK293 cells (embryonic neuronal tumor from a human female kidney) were grown for several days in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) with high glucose, 10% FBS, 100 mg/L penicillin–streptomycin (Invitrogen). They were harvested in PBS with 0.25% trypsin and 1 mM EDTA, washed with MEM, centrifuged (300 g, 10 min), and re-suspended in MEM. The human KCa3.1 gene (wild-type hKCa3.1) was subcloned into the expression vector, pCMV6-XL5 (OriGene, Rockville, MD). The plasmids, pCMV6-XL5-hKCa3.1 and pEF-GFP, were co-transfected using LipofectAMINE (Invitrogen) for 36 h according to the manufacturer’s protocol. HEK293 cells were plated at 5.5 × 10⁴ cells/coverslip for single-channel patch-clamp analysis.

Full length of S100A4 gene were inserted into a vector plasmid pCMV6-XL5 (OriGene Technologies). MCSC cells were transfected with pCMV6-XL5-S100A4 plasmid (pCMV-S100A4) performing in Lipofectamine™ 2000 (Invitrogen) following the manufacturer’s instructions, which were referred to as MCSCs/S100A4-vector. The expression of S100A4 was detected at 48 and 72 h after transfection on transcription and translation level respectively.
S100A4 was knockdown by S100A4-siRNA transfection. Double-stranded siRNAs specific to S100A4 were bought from Shanghai GenePharma Co., Ltd. (Shanghai, China). The S100A4 siRNA sequences were 5’-TGTAACGAATTCTTTGAAG-3′, and 5’-ACGAATTCTTTGAAGGCTT-3′. The non-coding (NC) siRNAs sequence was 5’-UUCUCCGAACGUGUCACGUTT-3′. MCSCs were transfected with a final concentration of 20 nM of siRNA performing in Lipofectamine™ 2000, which were referred to as MCSCs/S100A4-siRNA or MCSCs/NC-siRNA cells, respectively.