Nucleofector kit t

Human codon-optimized coding sequences of MERS-CoV nsp3-6 were designed using GeneArt, ordered from Thermo Fisher in four fragments, and subsequently assembled in low-copy-number vector pACNR1180 (64 (link)) using conventional cloning. The precise parts of MERS-CoV pp1a used for polyprotein constructs are outlined in Table S1 in the supplemental material. The nsp4 construct included the 21 C-terminal aa of nsp3 to prevent the N-terminal hydrophobic region of nsp4 from acting as a signal sequence, which could result in improper membrane insertion. In all constructs with a C-terminal myc or V5 tag, the C-terminal glutamine of the viral sequence was omitted to prevent the removal of the tag by M^pro. The SARS-CoV nsp3 gene (Frankfurt 1 strain, pp1a amino acids 819 to 2740) was synthesized by Bio Basic Inc. (Ontario, Canada). Coding sequences were transferred to the pCAGGS expression vector (Addgene) for expression. pCAGGS-SARS-nsp4 was described previously (42 (link)). 293T cells were transfected using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s instructions. HuH-7 cells were transfected using a Nucleofector 2b device (Lonza) with Nucleofector kit T (Lonza) in 6 × 10⁶ cells and 12 µg of plasmid DNA per transfection. Cotransfections were carried out with equimolar amounts of plasmids.

For both MyLa2000 and Hut-78 cells, transfection was carried out using Amaxa machine (Lonza, Basel, Switzerland) and Nucleofector Kit-T (Cat VCA-1002) according to manufacturer's instruction. Briefly, 10×10⁶ cells were centrifuged at 90g for 6 minutes and resuspended in 100 μl Nucleofector® Solution combined with siNRAs at room temperature. 50 nM small interfering RNA was used for the specific knockdown of USP2 (Santa Cruz Biotechnology, Texas, USA) and p53 (Thermo Scientific, Chicago, USA). Silencer select negative control #1 (Ambion Inc., Austin, TX, USA) was used as negative control. Cell/siRNA suspension was transferred into certified cuvette, and Nucleofector® Program T-016 was chose for MyLa2000 cells and V-001 was used for Hut-78 cells. Efficiency of transfection was evaluated at mRNA level by RT-PCR (Figure S1) and at protein level by western blot analysis.

Cells were seeded 24 hr. before transfection with 2 μg of negative control (pENTR221-β-glucuronidase), GATA2 (pENTR221-GATA2) or GFP vector using an Amaxa Nucleofector I Device. Nucleofector Kit T and Kit R (Lonza) were used to transfect NALM-6 and REH respectively. At 72 hr. samples were taken for Q-PCR or western blot. REH cells were transfected with 30 nM pre-miR-650 (Ambion) or FAM-labeled Pre-mir Negative Control #1 (Applied Biosystems). Identical methods were used to transfect with miR-362 or control htr vectors [48 (link)] obtained from the Human miRNA Library (Source BioScience).

EPAC1 was knocked down by siRNA in the ALL cell line NALM-6. To this end, a total of 3 × 10⁶ NALM-6 cells were transfected with small-interfering RNA (siRNA) by using a nucleofector device (Amaxa Biosciences) and the Nucleofector Kit T (Lonza, #VVCA-1002) according to the manufacturer’s instructions and using the program C-005. For the knockdown of EPAC1, we used 53 nM of ON-TARGETplus Human RAPGEF3 siRNA SMARTPool (L-007676-00-0010), which is a cocktail of four different siRNAs with the target sequences: CGUGGGAACUCAUGAGAUG, GGACCGAGAUGCCCAAUUC, GAGCGUCUCUUUGUUGUCA, CGUGGUACAUUAUCUGGAA. Equimolar concentration of a non-targeting siRNA (D-001810-01-05) was used as control. All siRNAs were obtained from Dharmacon. After transfection, the cells were incubated for 72 h before further treatments were initiated.

HEK293T cells were grown overnight in Opti‐MEM Reduced Serum media with 5% FBS (51985‐034, Thermo Fisher). When 90% confluent, cells were transfected using Lipofectamine 3000 reagent (L3000008, Life Technologies) according to the manufacturer's recommendations. K562 cells were nucleofected using a Nucleofector™ 2b Device (Lonza) using nucleofector kit T (VACA‐1002, Lonza) and protocol T‐016.