Pires2 egfp

Whole wild-type and mutant SIGMAR1-ORFs were distinctively amplified, with the use of appropriate restriction sites containing primers (online supplementary table S1), from cDNAs of a homozygous healthy individual and a patient with HMNJ, respectively and cloned into pEGFP-C3 (BD, California, USA) and pFLAG-CMV-2 (Sigma-Aldrich, Missouri, USA) vectors. With the employment of a Flag-tag containing forward primer and an appropriate reverse primer, Flag-tagged SIGMAR1 whole-ORFs were cloned from pFLAG-CMV-2 to the bicistronic vector pIRES2-EGFP (BD). All of the plasmid constructs were sequenced and verified to be free of any nucleotide changes in the SIGMAR1 cDNA.

The full-length cDNA of mPRα was amplified using the following primers: 5′-CATGGCGACGGTGGTGATG-3′ (forward) and 5′-GGCAGCAGAAGAAATAGGCG-3′ (reverse), and then subcloned into the vector, pIRES2-EGFP (BD Biosciences Clontech). The sequence for the construction of mPRα-shRNA was sense, 5′-GGAGCTGTAAGGTCTTCTTTA-3′; and antisense, 5′-TAAAGAAGACCTTACAGCTCC-3′, and then subcloned into the vector, pSUPER (GenerayBiotech Co., Ltd., Shanghai, China). Sequence fidelity and reading frame accuracy of the mPRα expression or mPRα-shRNA plasmid were achieved by DNA sequencing analysis. The MB231 or MB231br cells were transfected using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with the mPRα expression plasmid or mPRα-shRNA plasmid, respectively. The pIRES2-EGFP vector or pSUPER vector was also transfected into the MB231 or MB231br cells which served as controls. The transfected cells were then cultured in medium supplemented with G418 (Promega, Madison, WI, USA) or puromycin (InvivoGen, San Diego, CA, USA) for the establishment of stably transfected cell clones.