Lip2000 transfection reagent

PK-15 cells grown to 60–70% confluence in 12-well cell culture plates were transfected with GRP78 shRNA vector and pEGFP-GRP78 eukaryotic expression plasmid using the lip2000 transfection reagent (Invitrogen). Briefly, 1.5–2 μg of vector and 3.75–5 μl of lipofectamine 2000 were diluted in 100 μl Opti-MEM medium in 1.5 ml eppendorf tubes, respectively, stored at room temperature for 5 min, and then the two above-mentioned tubes were gently mixed and further incubated at room temperature for 20 min. The medium was removed and replaced with 1 ml of Opti-MEM containing the transfection mixture and further cultured at 37°C for 4 h, next, the supernatant was replaced with fresh maintenance medium and further incubated for 48 h. The gene knockdown and over-expression efficiency of GRP78 were evaluated by immunoblotting. The scramble shRNA vector and PEGFP-N₁ plasmid were used as negative controls, respectively.

Influenza virus A/environment/Qinghai/1/2008 (H5N1) was propagated in 9-day-old embryonated chicken eggs from specific-pathogen-free flocks (Beijing MERIAL Ltd.), at 37°C for 2 days. Allantoic fluid was clarified by centrifugation and stored at −80°C until use. Virus titer was determined by plaque assay. A549, MDCK, and HEK293T cells were routinely maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 μg/mL penicillin-streptomycin (HyClone, USA) and cultured at 37°C with 5% CO₂. Cells were transiently transfected with Lip2000 transfection reagent (Invitrogen, USA). The dual expression plasmid constructs pcDNA5/FRT/TO/HA-MEK1/IRES/myc-Ubc9 (HA-MEK1-Ubc9), pcDNA3.1-Flag-SUMO1 (SUMO2/3, SUMO4), pcDNA3.1-Myc-Ubc9, and pcDNA3.1-HA-MEK1 were constructed by add-on PCR by insertion between BamHI and XhoI sites of the pcDNA3.1(+) vector (Invitrogen). The MEK1^K64R mutant was generated by replacing lysine (K) 64 with arginine (R).

Plasmids expressing Cas9 and sgRNA were co-transfected into HepG2 cells using lip2000 transfection reagent (Invitrogen). At 36-h post-transfection, the cells were selected with puromycin at 2 μg/mL. After 2 days, the living cells were added to a 96-well plate at a cell density of 1 cell/well. Immunoblotting was performed with TRIM5γ-specific antibodies to ensure the gene knockout (KO) results and DNA sequencing was performed to further confirm the results of gene knockout. The sequences of the sgRNAs are listed in Supplementary Table 2.

LncRNA and mRNA silencers (si-AK001796#1, si-AK001796#2, si-AK001796#3 and
si-GAB1), miRNA mimics (miR-150 mimics), miRNA inhibitor (miR-150 inhibitor),
negative control (NC), and inhibitor negative control (NC inhibitor) were
designed by GenePharma (China). The predicted miRNA binding sites and mutations
of binding sites were designed and inserted into the pcDNA3.1 vector (Promega,
USA), which form the plasmid of AK001796-wt and AK001796-mut. Both of them were
purchased from Genecreat (China). All of them were transfected with Lip2000
Transfection reagent (Invitrogen, USA) with optimal concentration, 20 μM for
miRNA mimics, inhibitors, and siRNAs and 4 μg for plasmids. After transfection,
the cells were cultured at 37 ℃ for subsequent studies.

293A cells were purchased from HonorGene. A Dual Luciferase Assay Kit (E1910) was purchased from Promega. pHG-miRTarget-JAM3 WT-3U and pHG-miRTarget-JAM3 MUT-3U were transfected into the plasmid. The cells were cultured and transfected with Lip2000 transfection reagent (11668019, Invitrogen) to transfect all plasmids, hsa-miRNA NC, and hsa-miR-127-5p (GenePharma). The specific groups were as follows: miRNA NC+JAM3 WT group (cells cotransfected with phG-miRtarget-JAM3 WT-3U large plasmid and miRNA NC), hsa-miR-127-5p+JAM3 WT group (cells cotransfected with phG-miRtarget-JAM3 WT-3U large plasmid and hsa-miR-127-5p mimics), miRNA NC+JAM3 MUT group (cells cotransfected with pHG-miRTarget-YAP1 MUT-3U large plasmid and miRNA NC), and hsa-miR-127-5p+JAM3 MUT group (cells cotransfected with pHG-miRTarget-YAP1 MUT-3U large plasmid and hsa-miR-127-5p mimics). The cells were lysed with 1 × PLB lysate. Then, 20 μL of cell lysate was combined with 100 μL LARII solution and 100 μL Stop&Glo solution to detect firefly and sea kidney luciferase activity.