Lipofectamine messengermax

HepG2 (ATCC HB-8065) and BHK-21 (ATCC CCL-10) cells, purchased from the American Type Culture Collection (Manassas, VA, United States), were grown in RPMI (Gibco BRL) supplemented with 10% FBS, 2 mM glutamine, 100 μg/ml streptomycin and 100 U/mL penicillin. After 24 h incubation, cells were transfected using Lipofectamine MessengerMax according to the protocol provided by the manufacturer (ThermoFisher, Plainville, MA, United States). Briefly, cells were seeded into 12-well tissue culture plates and transfected with 1 μg MmPah mRNA using 2 μl Lipofectamine. EGFP mRNA was used as a positive transfection control in each experiment. To collect PAH for enzymatic activity assessment, 100 μl RIPA buffer (ThermoFisher) with Halt™ Protease Inhibitor Cocktail (ThermoFisher) were added to each well. Cell lysates were collected and stored at −80°C for further analysis.

CCTβ in U2OS and Huh7 was disrupted according to the standard protocol^{66 (link)}. Nucleotide sequences (467–486 for U2OS and 429–448 for Huh7) in PCYT1B mRNA (Accession No. NM_004845.5) that are commonly used for CCTβ2 and CCTβ3 were selected using web-based prediction software (Crispr direct; http://crispr.dbcls.jp)^{67 (link)} and transcribed with a MEGAshortscript T7 Transcription Kit (Thermo Fisher). Cells were co-transfected with the in vitro-transcribed guide RNA and GeneArt CRISPR Nuclease mRNA (Thermo Fisher) using Lipofectamine Messenger MAX (Thermo Fisher) and examined for genome digestion by GeneArt Genomic Cleavage Detection Kit (Thermo Fisher) 2 days after transfection. Clones obtained by single-cell cloning were checked by DNA sequencing to confirm genome editing and western blotting.

Lipofectamine MessengerMAX (ThermoFisher Scientific, Waltham, CA, USA) was used as a control. According to the manufacturers’ protocol, 1.5 μL of Lipofectamine Messenger MAX was incubated with 1000 ng of GFP-encoding mRNA for 10 min.

In vitro transcription for WT (T7-KLF2), ATG-mutated KLF2 (T7-mock), TXNDC5, and cysteine mutant TXNDC5 mRNA transcripts was performed using the mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher Scientific, MA, USA). HAEC were transfected with KLF2, KLF2-mock, TXNDC5, and cysteine-mutated TXNDC5 mRNA transcripts using the Lipofectamine MessengerMAX (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions.

The third-generation primary osteoblast-like cells were seeded in 24-well plates at a density of 4 × 10⁴ cells/well. Gluc mRNA (1 μg/well) was transfected using Lipofectamine MessengerMax (Thermo Fisher Scientific, Waltham, MA, USA). Gluc proteins secreted in the culture medium were measured using Renilla-Glo™ Luciferase Assay Reagent (Promega Co. Madison, WI, USA).

The maximum transfection efficiency with Lipofectamine MessengerMax (mMax; Thermo Scientific) was obtained with serum free condition (data not shown). Cells were passaged on 6-well plate on day 0 and cultured to 50 or 100% confluency on day 1. Cells were washed once with DMEM (no antibiotics and no serum) and then added DMEM (1 ml/well, 6-well) prior to transfection. For serum-free srRNA transfection, we used B18R mRNA to suppress the IFN responses and omitted the 20% B18R-CM treatment. Transfection complex with 5F-srRNA (1 μg), B18R mRNA (1 μg) and mMax reagent (6 μl) for one well of 6-well were prepared according to the manufacture’s protocol except for omitting the 10 min incubation of diluted messengerMax reagent and transfected into cells in the absence of serum. No incubation of the diluted messengerMax is significantly affect on the transfection efficiency of srRNAs. After 3 hr incubation, medium was changed to the Advanced DMEM containing 20% B18R-CM. 4 hr incubation will increase transfection efficiency for mMax reagent in current protocol (data not shown). For puromycin-free condition, cells were passaged to Matrigel or STO feeder on day 3 or day 5 with 6 or 12 times dilutions. ES culture medium was used from day 5.