Lipod293 in vitro dna transfection reagent

Plasmid transfections (vector, HA-RNMT, FLAG-RAM, CBP-MYC) were performed using LipoD293™ In Vitro DNA Transfection Reagent per protocol (SignaGen). Cells were harvested for protein or RNA 24 h post-transfection. Silencer Select Pre-designed siRNAs were obtained from Life Technologies and transfected using RNAiMax (Invitrogen; 20nM). Cells were harvested for protein or RNA 48 h post-transfection. Silencer Select negative control #2 (Life Technologies) was used as a transfection control.

For T-cell factor (TCF) reporter assays, low-passage cells (10⁵) were plated onto six-well plates. One day following plating, each well was transfected with 0.9 μg 14X Super TOP or 8X FOP FLASH plasmid (33 (link)) and 0.09 μg pRL-SV40 using LipoD293™ In Vitro DNA Transfection Reagent per protocol (SignaGen). Two days following transfection, cells were lysed, and luciferase and Renilla activity were measured with Dual-Glo luciferase reagents (Promega). Luciferase readings were normalized against Renilla readings, and TOP FLASH/FOP FLASH ratios were calculated.

293FT cells (ThermoFisher Scientific, Cat# R70007) were used to
generate lentiviral particles through co-transfection of the packaging
vectors pCMV-dR8.2 dvpr (Addgene, Plasmid # 8455) and pCl-VSVG (Addgene,
Plasmid # 1733) with the appropriate overexpression, shRNA, or other
plasmid. 293FT cells were seeded at a density of 1.2 million cells in DMEM,
high glucose (ThermoFisher Scientific, Cat# 11995073) in 10% Fetal Bovine
Serum (ThermoFisher Scientific, Cat# 26140079 with 1%
Penicillin-Streptomycin (ThermoFisher Scientific, Cat # 15140122). Cells
were incubated for 24 hours prior to transfection. Transfection was
performed using LipoD293^™ In Vitro DNA Transfection
Reagent (SignaGen Laboratories, Cat # SL100668) according to the
manufacturer’s instructions. Briefly, 5μg each of the
packaging plasmids and the plasmid of interest were combined into a tube,
the transfection reagent was diluted and added followed by a 15 minute
incubation. The transfection mixture was then added to the 293FT cells.
Media was changed after 16 hours. Virus was collected 48 hours after media
change and concentrated using the Lenti-X Concentrator (Clontech Takara Bio
USA, Cat # 631232) according to the manufacturers instructions. Viral
supernatants were centrifuged at 1,500 x g for 45 minutes and viral pellets
were frozen at −80°C for future use.

Cell lines were purchased from the American Type Culture Collection or Deutsche Sammlung von Mikroorganismen und Zellkulturen, and their genotypes were confirmed by short tandem repeat (STR) analysis (81 (link)). Cells were tested regularly for mycoplasma by using a PCR-based kit from Agilent Technologies. MDA-MB-453, BT474, SKBR3, UACC893, and 293T cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin (Pen/Strep). MDA-MB-361 cells were maintained in DMEM supplemented with 20% FBS and Pen/Strep. HCC1569, HCC202, HCC1419, and EFM192A cells were maintained in RPMI supplemented with 10% FBS and Pen/Strep. SUM225 cells were maintained in Ham’s F12 supplemented with 5% FBS, 5 μg/ml insulin, 1 μg/ml hydrocortisone, and 10 mM HEPES. Transient transfections of plasmids were performed by using LipoD293™ In Vitro DNA Transfection Reagent (SignaGen Laboratories). Transient transfections of SMARTPool SGK3 siRNA (L-004162–00-0005, Horizon Discovery) were performed with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific).

HEK 293T cells (ATCC CRL-3216, Lot # 62729596) were maintained in high
glucose DMEM supplemented with 10% of fetal bovine serum (Life Technologies) and
GlutaMAX (Gibco). PrP^C knockout (PrPKO) N2a cells have been described
previously.^{52 (link)} PrPKO
N2a cells were maintained in low glucose DMEM supplemented with nonessential
amino acids (Corning), 10% of heat-inactivated fetal bovine serum (Life
Technologies), GlutaMAX, and MycoZap Plus-CL (Lonza). HEK 293T and PrPKO N2a
cell lines used in this study were mycoplasma free and were maintained at
37°C in a 5% of carbon dioxide incubator.
HEK 293T cells and PrPKO N2a cells used for western blotting were
transiently transfected using LipoD293 In Vitro DNA Transfection Reagent
(SignaGEN Laboratories) with PrP^C encoding pcDNA3.1(+)Hygro plasmids
in 6-well plates. Fifteen to eighteen hours after cells were transfected, the
media was changed and cells were allowed to recover for 24 hours. PrPKO N2a
cells used for electrophysiology were transiently transfected using
Lipofectamine 2000 with pEGFP-N1 (Clontech).