Lipofectamine 3000 transfection

HEK293T cells were seeded in 12-well plates and transfected with AVPR1A-hRluc alone or together with plasmids encoding EYFP or ACKR3-EYFP using the Lipofectamine 3000 transfection reagent (ThermoScientific). For BRET titration assays, AVPR1A-hRluc at the fixed amount of 50 ng was co-transfected with increasing amounts of EYFP or ACKR3-EYFP. For BRET assays at a constant acceptor : donor ratio, increasing amounts of AVPR1A-hRluc and ACKR3-EYFP were co-transfected at a ratio of 1 : 10. In all assays, empty vector pcDNA3 was added to maintain the total cDNA amount for each transfection reaction constant. After an overnight incubation, cells were seeded in poly-l-lysine coated 96-well white plates and incubated again overnight. Cells were then washed with PBS and fluorescence was measured in a Biotek Synergy II plate reader (λ_excitation 485 nm, λ_emission 528 nm). For BRET measurements, coelenterazine H (Nanolight Technology) at 5 µM in PBS was added to the cells. After 10 min incubation at room temperature, luminescence was measured at 460 ± 40 and 528 ± 20 nm. The BRET signal is calculated as the ratio of RLU measured at 528 ± 20 nm over RLU at 460 ± 40 nm. The net BRET is calculated by subtracting the BRET signal detected when the AVPR1A-hRLuc was transfected alone. For titration experiments, net BRET ratios are expressed as a function of EYFP/total luminescence.

A TCF/LEF reporter assay was used to examine the effect of Hcy/HTL on WNT‐induced transcriptional activity. First, 2.5 μg TOPFLASH (gifted from Dr. Tao Zhong's lab, Fudan University (Ni et al, 2011)) and 2.5 μg FOPFLASH plasmids (Millipore, Bedford, MA, USA; Appendix Table S6) were transfected in 1 × 10⁶ NE4C cells using Lipofectamine^® 3000 transfection reagent (L30075, Thermo Fisher, Waltham, MA, USA) for 48 h. Hcy/HTL was added in the culture medium 4 h before cell collection. Reporter activities were measured with a plate reader (LD400; Beckman Coulter, Fullerton, CA, USA) using a dual‐luciferase reporter assay system (Promega, Madison, WI, USA). TCF/LEF activity was defined as the ratio of TOPFLASH:FOPFLASH reporter activities.

MCF7 shFTH1 and H460 shFTH1 cells were seeded in six-well plates at 3 × 10⁵ cells/well and grown overnight prior to transfection.
All plasmids were transfected with Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) following manufacturer’s instructions. FTH1 reconstitution was performed using 2.5 μg/μl of the expression vector containing the coding sequence of human FTH1 cDNA (pcDNA3/FTH1) (MCF-7 shFTH1/pcDNA₃FTH1 and H460 shFTH1/pcDNA₃FTH1), while 2.5 μg/μl of pcDNA₃ plasmid was used as negative control (MCF-7 shFTH1/pcDNA₃ and H460 shFTH1/pcDNA₃). Transfection efficiency was tested by western blot and qPCR after 48 hrs. All transfection experiments were repeated in triplicate.