Hygromycin b

The pSecTag2A/Hygro expression plasmid (Invitrogen) encoding myc-tagged human soluble TWEAK was constructed and both the vector alone and this plasmid were transfected into B16 cells as described [21 (link)]. Stable B16 transfectants were selected using 400 μg/ml hygromycin B (Corning) and individual clones were screened for TWEAK expression by Western blot analysis using a myc antibody (see below). One control (vector-transfected) and one TWEAK overexpressing cell line were chosen for subsequent analysis (designated V2 and T2, respectively). The pcDNA6/His expression plasmid (Invitrogen) encoding myc-tagged human soluble TWEAK was constructed and transfected in a similar manner as above, but in this case stable vector- or TWEAK plasmid-transfected B16 cells were selected using 10 μg/ml blasticidin (Sigma-Aldrich). Following Western blot screening of individual cell clones, one control (vector-transfected) and one TWEAK overexpressing cell line was chosen for subsequent analysis (designated V1 and T1, respectively).

HEK293T cells, Vero cells, BHK cells and HUVEC were purchased from the American Type Culture Collection (ATCC). EA.hy926 cells were purchased from the tissue culture facility at the University of North Carolina at Chapel Hill. 293FT cells were purchased from Thermo Fisher. iSLK.219 cells were a kind gift from Don Ganem. HEK293T cells, Vero cells, BHK cells, 293FT cells, and EA.hy926 cells were maintained in Dulbecco’s minimal essential medium (DMEM) (Corning) containing 10% fetal bovine serum (FBS) (Sigma) and 1% penicillin-streptomycin (Pen-Strep) (Corning). HUVEC were maintained in EGM-2 supplied with growth factors obtained from the EGM-2 Bullet kit (Lonza). iSLK.219 cells were maintained in DMEM containing 10% tetracycline (Tet)-free FBS (Sigma), 1% Pen-Strep, 10 μg/ml puromycin (Corning), 50 μg/ml Geneticin (Corning), and 100 μg/ml hygromycin B (Corning). All cells were maintained at 37°C in a 5% CO₂ laboratory incubator which was subject to routine cleaning and decontamination. HSV-1 (KOS strain) was obtained from ATCC and propagated in Vero cells. VSV was a kind gift from Doug Lyles and was propagated in BHK cells. KSHV was purified form iSLK.219 cells in our lab.

Purified, endotoxin-free plasmid DNA carrying different constructs in pTRE2hyg vector or its derivatives were transfected into CHO-K1 cells (Clontech) with FuGene HD transfection reagent (Promega, Madison, WI, USA, cat# E2311) according to the manufacturer. Two days post-transfection, selection of stably transfected cell lines was started in DMEM low glucose medium (Biosera, Nuaille, France, cat# LM-D1102/500) supplemented with 10% tetracycline-free fetal bovine serum (Biosera, cat# FB-1001T) and 250 µg/mL hygromycin B (Corning, NY, USA, cat# 30-240-CR). The selection was continued for 2–3 weeks with frequent changes in the selection medium until distinct colonies could be visualized. In order to minimize the integration site-dependent differences in the expression of the transgenes, several thousand independent stable transfected colonies were pooled and maintained as a stock cell line. Expression of the transgenes was induced with 1.5 µg/mL doxycycline (Clontech, cat# 631311). Mammalian cells were kept at 37 °C humidified incubator in the presence of 5% CO₂.

GHSR activity was examined using the Tango β-arrestin recruitment assay that was developed by B.L. Roth (89 (link)). HTLA cells stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene were a gift from B.L. Roth (University of North Carolina, Chapel Hill, North Carolina, USA). HTLA cells were maintained in DMEM with 10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin/100 μg/mL streptomycin (Thermo Fisher Scientific), 2 μg/mL puromycin (Tocris Bioscience), and 100 μg/mL hygromycin B (Corning) at 37°C with 5% CO₂. Cells were transfected with a GHSR-Tango plasmid expressing human GHSR and a vasopressin receptor (V₂) tail (66293, Addgene) using the calcium-phosphate method and maintained for 24 hours prior to the next step. Transfected cells were transferred into a poly-D-lysine–coated 96-well plate (Corning) and maintained for 24 hours before treatment. Cells were then treated with an appropriate amount of human ghrelin peptide (Phoenix Pharmaceuticals, 031-30), LEAP2 peptide (Phoenix Pharmaceuticals, 075-40), or combinations of the 2 (LEAP2 was added 2 hours prior to ghrelin) at different ratios and doses for 24 hours to allow the expression of luciferase. Treated cells were incubated with Bright-Glo Luciferase Assay solution (Promega, E2610), and the signal was detected using a Biotek Neo2 microplate reader.