Pcmv3 plasmids

Primary antibodies were provided by following sources: DPP4, DHCR24, BrdU, and β-Actin (Abcam, Cambridge, MA, USA); Methotrexate (MTX) and DPP4 inhibitor Sitagliptin were obtained from Selleckchem (Houston, TX, USA). pCMV3 plasmids encoding empty vector (EV) or DCHR24 were obtained from Sinobiological Inc. (Beijing, China). Lentiviral shRNA plasmids encoding scramble control, shDHCR24, and shDPP4 were provided by Genecopoeia Inc. Human GTN cell lines JEG3 JAR, and BEWO were provided by American Type Culture Collection (Manassas, VA, USA). These GTN cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 4 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin.

The pcDNA3.1 plasmids harboring the long form BMPR-2 and its kinase dead mutant BMPR-2^K230R were kindly provided by Dr. Petra Knaus at Freie University Berlin (Berlin, Germany). The following plasmids were used in the current study: pCDNA3 plasmids harboring ALK2^WT, ALK3^WT and ALK6^WT were purchased from Addgene (#80870, #80873 and #80882). pCMV3 plasmids harboring activin A receptor type 2A (ActR2A) and human activin A receptor type 2B (ActR2B) were obtained from Sino Biological Inc. The point mutations in human ALK2 (ALK2^R206H), human ActR2A (ActR2A^K219R) and ActR2B (ActR2B^K217R) were made in-house using the QuikChange II site directed mutagenesis method. The sequences for mutagenic oligonucleotides are shown in Supplementary Table S1. The mutations were confirmed by DNA sequencing.