Transfast transfection reagent

H1299 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) from Sigma Aldrich (Vienna, Austria), A549 and Calu-3 cells in a 1:1 mixture of Ham‘s F12 and DMEM. Media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 1.25 μg/ml amphotericin B (Gibco, Lifetechnologies; Vienna, Austria). To verify the origin of the various cell lines, cell lysates were sent to Microsynth (Balgach, Switzerland) for Sanger sequencing. Cells were transiently transfected at a confluence of 60 - 80% with 1.5 μg plasmid DNA encoding UCP2 or PRMT1 as well as with 100 μM siRNA UCP2 or PRMT1 using 2.5 μl of TransFast^TM transfection reagent (Promega; Madison, WI, US) in 1 ml of serum- and antibiotic-free medium. Transfection mix was replaced by full culture medium after 24 hours. All experiments were performed 48 hours after transfection. siRNAs were obtained from Microsynth, and their sequences (5′-3′) were as follows: UCP2
5′-CACTGTCGACGCCTACAAGACCATC-3′, ‘5′-GTCATAGGTCACCAGCTCAGCACAG-3′; PRMT1: 5′-TGCTCAACACCGTGCTCTATGC-3′, 5′-TCCTCGATGGCCGTCACATACA-3′.

Human G9a expression vector pWPXL-G9a-Myc-DDK was constructed and provided by Dr. Min-Wei Chen. To construct pWPXL-G9a-Myc-DDK, the sequence encoding G9a-Myc-DDK was excised from pCMV6-EHMT2-Myc-DDK (Cat. No.: RC219200, Origen) and inserted into pWPXL downstream of the EF1α promoter. HeLa, SiHa and CaSki cells were transient transfected with 5 μg of pWPXL-G9a-Myc-DDK or empty vector using the TransFastTM transfection reagent (Promega Corporation, Madison, WI) according to manufacturer’s instructions.

HeLa cells at passage >50 were cultured on glass cover slips (Ø = 30 mm) using DMEM (Sigma-Aldrich, Vienna, Austria) containing 10% FCS (PAA, Pasching, Austria), penicillin (100 U/ml) and streptomycin (100 U/ml) in a humidified incubator (37 °C, 5% CO₂/95% air). 2–3 days prior experiments HeLa cells were transfected in DMEM (without FCS and antibiotics) with plasmids (1–4 μg/ml total) and/or siRNA (100 nM) using 4 μg/ml TransFast^TM transfection reagent (Promega, Madison, WI, USA).

Transfection experiments were carried out following the method of Muniraju and colleagues (2015). Briefly, Vero or VDS cells (~70% confluent) grown in 6-well plates were infected with a recombinant fowlpox virus that expressed the T7-RNA polymerase at an MOI of 0.2 for 1 h. The cells were washed and transfected with an appropriate full-length PPRV plasmid and helper plasmids, pN, pP, and pL, using the TransFastTM transfection reagent (Promega, Madison, WI, USA) at a ratio of 6:1 (wt/wt) in a total volume of 0.75 mL of OPTI-MEM I reduced serum medium/well (Gibco, Waltham, MA, USA). The cells were observed daily under a microscope for the appearance of PPRV-specific CPE. Rescued viruses were further passaged at least three times before stocks of viruses were prepared for further study.

Transfection of EA.hy926 cells with a pool of 3 target-specific TRPV1 siRNA (VR1 siRNA; sc36826; Santa Cruz, CA, USA), was performed using Transfast^TM transfection reagent (Promega, Madison, WI, USA) according to the manufacturer's protocol. All experiments were performed 48 hours after transfection. The efficiency of siRNAs and of an appropriate negative control was approved by real-time, quantitative PCR and western blot.

HeLa cells were grown in Dulbeccos's Modified Eagle Medium (Sigma Aldrich) containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin, and they were plated on 30-mm glass coverslips. At 60–80% confluence, cells were transfected with 1.5 µg (per 30-mm well) of plasmid DNA encoding the appropriate sensor/fluorophore using TransFast^TM transfection reagent at 3 µg/well (Promega, Madison, WI) in 1 ml of serum- and antibiotic-free medium. Cells were maintained in a humidified incubator (37°C, 5% CO₂, 95% air) for 16–20 hours prior to changing the culture medium. All experiments were performed either 24 hours or 48 hours after transfection.