Pegfp n1 expression vector

To evaluate the pathogenic potential of the TMEM43 mutations, we cloned the wild-type human TMEM43 and TMEM43 missense mutation (p.S358L) into expression vectors [2] (link). PCR was performed using the clone TMEM43-IRAU13-C10 containing full length human TMEM43, forward primer 5′- GATGCTAGCATGGCCGCGAATTATTCCAG-3′ and reverse primer 5′-GCAGAATTCGCTCCAACTTTTTGGCTGGCAC-3′. The resulting PCR products were directionally cloned by EcoRI and NheI sites in pEGFP-N1 expression vector (Clontech, Mountain View, CA, USA) to generate plasmid p-h-TMEM43, which contains cDNA tagged with green fluorescent protein (GFP) at the C-terminus. Mutated p-h-mTMEM43 was obtained by site directed mutagenesis of the wild-type construct by using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The following mutagenic primers were used: 5′-CTGTGTGGCCACCTTGCTGACCCTGCT-3′ and antisense 5′-AGCAGGGTCAGCAAGGTGGCCACACAG-3′. The plasmids were verified by sequence analysis.

The human pancreatic cancer cell lines PANC-1, MIA PaCa-2 and the embryonic kidney cell line HEK293 were obtained from American Type Culture Collection and were cultured in DMEM (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT). CD147 pLKO.1 lentiviral shRNA (A6) was obtained from Open Biosystems. The MISSION® Non-Target shRNA Control Vector (pLKO.1-NTC) was obtained from Sigma-Aldrich. Human CD147 cDNA was subcloned into the pEGFP-N1 expression vector (Clontech, Mountain View, CA) as described previously [24 (link)].

DNA segments encoding the extracellular domain of human endoglin from amino acid Met1 (position 419) to Gly586 (position 2176) were generated by PCR using PfuTurbo DNA polymerase (Stratagene, La Jolla, CA, USA) and cloned into the pEGFP–N1 expression vector (Clontech, Takara Bio Europe SAS, Saint-Germain-en-Laye, France) inframe with the EGFP protein from Aequorea victoria (Figure 1). Briefly, PCR was carried out using pcEXV-ENG [32 (link)] as a template to generate the extracellular domain and in the presence of sequence-specific oligonucleotides with add-on sequences surrounded by NheI and HindIII sites (5′-ATA GCT AGC ATG GAC CGC GGC ACG CTC C -3′and 5′-CGC AAG CTT GCC TTT GCT TGT GCA ACC AGA -3′, respectively). The resulting fragments were double-digested with NheI and HindIII, and inserted at the NheI/HindIII sites of the pEGFP–N1 expression vector (Figure 1). The construct (pEGFP–N1/Eng.EC) was verified by DNA sequence analysis. For protein purification purposes, a streptavidin tag (Strep-tag^®II; WSHPQFEK) was added to the C terminus (GenScript Biotech, Leiden, The Netherlands) leading to the EGFP–N1–EndoglinCDS1 plasmid. In addition, plasmid pcDNA3.1 (ThermoFisher Scientific, Waltham, MA, USA), encoding the neomycin resistance gene was used as a negative control in cell transfections.

Full-length human ADM cDNA was amplified by PCR and cloned into the pEGFP-N1 expression vector (Clontech, Palo Alto, CA, USA) to construct pEGFP-N1-ADM, and then transfected into HuCCT1 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells transfected with pEGFP-N1 were used as a negative control. Stable ADM-expressing clones were selected using geneticin (Roche Diagnostics, Indianapolis, IN, USA) at a concentration of 500 µg/ml.

A cDNA fragment containing full‐length FBN1 (GenBank ID: NM_000138.4) derived from human muscle cDNA and suitable restriction sites was PCR‐amplified using KOD‐Plus‐Neo (Toyobo). The PCR amplicons were cloned into the NheI and SacII sites of the pEGFP‐N1 expression vector (Clontech, Takara Bio). Mutant EGFP‐FBN1 plasmids were generated by site‐directed mutagenesis and their construction is confirmed by direct Sanger dideoxy sequencing. Gly2003Arg (G2003R) which was previously reported in an adolescent idiopathic scoliosis (AIS) case was used as a positive control (Buchan et al., 2014). Mutations of p.Tyr2596Thrfs*86 (Y2596Tfs*86), p.Glu2759Cysfs*9 (E2759Cfs*9) and Gly2003Arg were introduced into a wild‐type (WT) pEGFP‐FBN1 QuikChange Lightning Site‐directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer's instructions. The resulting three mutant plasmids pEGFP‐FBN1‐Tyr2596Thrfs*86, pEGFP‐FBN1‐Glu2759Cysfs*9, and pEGFP‐FBN1‐Gly2003Arg were used for functional studies.