For EZH2 overexpression, the EZH2 gene was subcloned into the pEZ-M98 vector (GeneCopoeia, Inc.) to generate pM-EZH2. Cultured N-PASMCs were then transfected with pM-EZH2 vector using the Neon Transfection System (MPK5000) from Invitrogen (Thermo Fisher Scientific, Inc.), according to the supplier's instructions. Briefly, cells were cultured in 60-mm dishes to 70% confluency and then transfected with 2 µg plasmid DNA (1,375 V; 20 ms) at room temperature. Empty vector pEZ-M98 (pM) was used as a transfection control.
For EZH2 knockdown, PASMCs from CTEPH patient 3 were cultured in 60-mm dishes and transfected with 160 pmol small interfering (si)RNA (Shanghai GenePharma Co., Ltd.) and treated with Lipofectamine RNAiMAX reagent from Invitrogen (Thermo Fisher Scientific, Inc.) at room temperature, according to the manufacturer's instructions. The sequences were sense, 5′-CCUGACCUCUGUCUUACUUTT-3′ and antisense, 5′-AAGUAAGACAGAGGUCAGGTT-3′. RNAi negative control that had no homology with mammalian genes was used as the transfection control. The RNAi negative control sequences were sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. After 48 h, transfection efficiency was determined by both PCR and western blot analysis for EZH2.
For EZH2 knockdown, PASMCs from CTEPH patient 3 were cultured in 60-mm dishes and transfected with 160 pmol small interfering (si)RNA (Shanghai GenePharma Co., Ltd.) and treated with Lipofectamine RNAiMAX reagent from Invitrogen (Thermo Fisher Scientific, Inc.) at room temperature, according to the manufacturer's instructions. The sequences were sense, 5′-CCUGACCUCUGUCUUACUUTT-3′ and antisense, 5′-AAGUAAGACAGAGGUCAGGTT-3′. RNAi negative control that had no homology with mammalian genes was used as the transfection control. The RNAi negative control sequences were sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. After 48 h, transfection efficiency was determined by both PCR and western blot analysis for EZH2.