Lipofectamine messengermax transfection reagent

Total RNA was extracted as before and transfected using Lipofectamine MessengerMAX transfection reagent as recommended by the manufacturer (InVitrogen); one microgram of RNA was used for 7.5 × 10⁴ cells seeded in a well of a 12-well plate. In some experiments, oligodT affinity purification was used, as recommended by the supplier (Invitrogen™ Dynabeads™ mRNA Purification Kit Catalog No. 61006), to separate polyadenylated RNA from non-polyadenylated RNA. Polyadenylated RNA was recovered by elution while non-polyadenylated RNA in the flowthrough was left to precipitate overnight at −20 °C after addition of 3 volumes of ethanol, before being resuspended in ultrapure water. Approximately 95% of total RNA was recovered in this fraction. For transfection, the same fraction of either polyadenylated or non-polyadenylated RNA was used, transfected into L929 cells using Lipofectamine MessengerMAX transfection reagent as recommended by the manufacturer (InVitrogen). In each case, one microgram of RNA was used; for polyadenylated RNA, 50 nanograms was used and completed to one microgram with non-polyadenylated RNA from mock-infected cells. Transfected cells were recovered 8 h post-transfection.

Chemically synthesized Dicer-Substrate Short Interfering RNA (DsiRNA) [33 (link)] targeted to exon 3 was used for knockdown of the SDHB gene. The control and SDHB-targeted DsiRNAs (Table 1) were transfected in CAMKK2^−/− HEK293 cells using Lipofectamine™ MessengerMAX™ Transfection Reagent (ThermoFisher Scientific, Cat. No: LMRNA001). The transfected cells were cultured for 48 h and then harvested for ER/mitochondrial enrichment, SDH enzyme activity measurement, and Western blotting. The full-length SDHB open reading frame (ORF: Accession number: NM_0030000.3) was chemically synthesized in the pcDNA3.1(+)N-DYK vector (GenScript: clone identification number: OHu18105C) and transfected in CAMKK2^−/− HepG2 cells using Lipofectamine™ 3000 Transfection Reagent (ThermoFisher Scientific, Cat. No: L3000001). The transfected cells were selected by adding 800 μg/mL G-418 (Sigma, Cat. NO: 4727878001) to the cell culture medium, and a pool of SDHB overexpressed cell populations were harvested after two weeks of culture. Subsequently, the SDHB overexpressed cells were expanded and used for ER/mitochondrial enrichment, SDH enzyme activity measurement, and Western blotting.

HeLa and HEK293 cells were obtained from ATCC (Manassas, VA); and human hepatic stellate cells from Creative Bioarray (Shirley, NY). Monolayer cell culture was prepared per supplier instructions for mRNA transfection. Briefly, cells with 80% confluency were transfected with 2.5 µg of each mRNA and 4 uL Lipofectamine™ MessengerMAX™ transfection reagent (Thermo Fisher Scientific, Inc., Waltham, MA). Cell lysates and culture media were collected at the indicated time points for capillary gel electrophoresis or SDS-PAGE followed by immunoblotting with anti-hADAMTS13 (rabbit mAb ab177940, 1:2,000) or anti-GAPDH (mouse mAb ab125247, 1:6,000, both from Abcam, Cambridge, MA) and detection with the corresponding secondary antibodies. The collected culture media were also subjected to quantitative measurements of rhADAMTS13 as below.