Lipofectamine rnaimax transfection

A total of 2 × 10⁵ A549 cells were seeded into 12-well plates (Sigma-Aldrich) and then transfected with siRNA targeting TRAPPC6AΔ (5′-CUGUGUUGUUUGAGUUUCU-3′) or nontargeting siRNA (Genepharma, Shanghai, China) at a concentration of 40 nM for 48 h by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's instructions. The knockdown efficiency was checked by Western blotting. To study the effect of TRAPPC6AΔ knockdown on the growth of influenza virus, the WSN virus was used to infect siRNA-treated A549 cells at an MOI of 0.01. Supernatants were collected at 24 and 48 h p.i., and virus titers were determined by plaque assays on MDCK cells.

The oligonucleotide sequences targeting GCN5 and CHIP mRNA were as follows: GCN5: #1 CCAACTGTCGCGAGTACAA, #2 GAAGCTGATTGAGCGCAAA; CHIP: TGCCGCCACTATCTGTGTA.
Transfection was performed according to the manufacturer’s instructions using Lipofectamine RNAi MAX transfection reagent (Invitrogen) and 50 nM siRNA.

U2OS parental cells were plated on 100-mm plates. The next day, cells were transfected using 10 μM siRNA listed at the end of this paragraph. Ten μM MISSION siRNA Universal Negative Control (Sigma-Aldrich, cat no. SIC001) was used as a “non-targeting” control in our experiments. Lipofectamine RNAiMAX transfection (Invitrogen; cat no. 13778-100) protocol was used in the siRNA knockdown. At 48 h post-transfection, the cells were harvested and knockdown confirmed by immunoblotting for the protein of interest. Segregation assays were performed as described previously, after treating the cells with the siRNA of interest on day 3 of the protocol. All siRNAs were purchased from Sigma-Aldrich: siRNA TopBP1-A CUCACCUUAUUGCAGGAGAdTdT; TopBP1-B GUAAAUAUCUGAAGCUGUAdTdT, CK2α-A GGCUCGAAUGGGUUCAUCUtt; CK2α-B GAUGACUACCAGCUGGUUCdTdT, CK2α’-A CAGUCUGAGGAGCCGCGAGdTdT, and CK2α’-B AUACAGCCCAAACUCCACAUUU.