Plenti vector

Lentivirus construction and infection were performed as formerly introduced (24 (link)). In brief, after digestion with EcoR I and Xba I (Takara, Dalian, China), pre-miR-486-5p sequence was cloned into pLenti vector (Invitrogen, Carlsbad, CA, USA) (named pLenti-miR-486). Viral particles containing pLenti or pLenti-miR-486 were collected to infect A549 and H1299 cells and further sorted with flow cytometry.
The wild-type (WT) RSK and p70S6K 3’UTR fragments and their corresponding mutant (Mut) fragments were cloned into pGL3-basic (pGL3, Promega, Madison, WI, USA) luciferase reporter vector to confirm their direct binding with miR-486-5p. These pGL3-derived vectors and miR-486-5p mimics were then cotransfected into 293T cells, and the Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA) was applied for luciferase activity measurement as previously described (25 (link)).
The sequences of RSK and p70S6K were constructed into the pcDNA3.1 plasmid. siRNAs for RSK (siRSK #1-3) and p70S6K (sip70S6K #1-3) were synthesized by RIBOBIO (Guangzhou, China). All siRNA sequences were listed in Table S2. Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) was used for transient transfection. All the constructed vectors were sequenced (Sangon Biotech, Shanghai, China) and the primers are listed in Table S3.

The pre-miR-411 (96 bp) was amplified using H1299 genomic DNA and cloned into the pLenti vector (Invitrogen) after double digestion by BamH I and Xho I, forming pLenti-miR-411 and sequenced (Sangon Biotech, Shanghai, China). Lentiviral particles were collected at 48 and 72 h from HEK293T cells following co-transfection using pLenti or pLenti-miR-411 vector with packaging plasmids (psPAX2 and pMD2G) and saved at 4 °C. The long-term storage environment should be at − 80 °C.
Infection of H1299 and SPC-A1 cells were performed at 50% density for 5 h with pLenti or pLenti-miR-411 viral particles and green fluorescence positive cells were sorted by flow cytometry (Beckman Coulter, Inc., Brea, CA, USA) to establish pLenti/pLenti-miR-411 stably transfected H1299 and SPC-A1 cells (pLenti/pLenti-miR-411 H1299, and SPC-A1), followed by enlarge cultivation for further experiments.

Flag-tagged Nef, Nef(1 to 115), or Nef(1 to 115)-(L112A/Y115A) genes were constructed into the pLenti vector (Invitrogen). HEK293T cells were cotransfected with pLenti-Nef, pNef(1 to 115), and pNef(1 to 115)-(L112A/Y115A) plasmids or pLenti empty vectors and pLP1, pLP2, and pLP/VSVG plasmids (Invitrogen) using PEI transfection reagents. Culture medium containing lentiviruses was collected 2 days after the transfection. CD4⁺ Jurkat T cells were infected with lentiviruses containing Nef, Nef(1 to 115), Nef(1 to 115)-(L112A/Y115A), or an empty vector. Stable Nef-, Nef(1 to 115)-, or Nef(1 to 115)-(L112A/Y115A)-expressing cells were selected with 2.5 μg/ml puromycin.

The full-length and truncated Nef were inserted into pCAGGS vectors (BCCM/LMBP Plasmid Collection, Ghent, Belgium) with a hemagglutinin (HA) tag linked to the C-terminal insert. Nef mutants were constructed into pCAGGS vectors using an overlapping PCR, and the putative amino acid residues were mutated to alanine. Alternatively, full-length Nef with the GFP gene fused to its N terminus was inserted into the pLenti vector (Invitrogen). Full-length and truncated LMP7 were inserted into pcDNA3.1 vectors (Invitrogen) with a 3× Flag tag linked to the N-terminal insert. Additionally, full-length LMP7 was inserted into the pCMV vector (Clontech, Fremont, CA, USA) with a Myc tag linked to the N-terminal insert. For the yeast two-hybrid assays, Nef was inserted into the pGBKT7 vector and LMP7 into the pGADT7 vector (Clontech). To generate pNL4-3dNef, the XhoI restriction enzyme site in pNL4-3 was digested, and a stop codon was inserted.

In brief, pri-miR-18a sequence was digested with BamH I and Xhol I, then cloned into the pLenti vector (Invitrogen, Carlsbad, CA, USA) and named pLenti-miR-18a. The CDS of IRF2 sequence were cloned into the pcDNA3.1(−) plasmid, formed pcDNA3.1-IRF2. The primer sequences used in this study are shown in Supplemantary Table 2.

A lentiviral system was used to construct cell lines with stable knockdown and overexpression. shRNAs targeting EHF, GLI1, and CCL2 were inserted into the PLKO.1 vector (Invitrogen) to construct knockdown plasmids. Plasmids was extracted by using a Steadypure Plasmid DNA Extraction Kit (Accurate Biotechnology (Hunan)Co., Ltd) following the manufacturer's instructions. A non‐specific scramble shRNA sequence was used as negative control. The shRNA sequences can be found in Table S2. EHF was also inserted into the plenti vector (Invitrogen) to construct overexpression plasmids. All the above plasmids were confirmed by DNA sequencing. Lentivirus‐infected stable cell lines were screened with puromycin (5 μg/mL) for 2 days.

Lentivirus construction and infection performed as previously described. In brief, pri-miR-199a sequence was digested with BamH I and Xhol I, which then cloned into the pLenti vector (Invitrogen, Carlsbad, CA, USA) to form pLenti-miR-199a. pLenti-miR-199a or pLenti vector was co-transfected into HEK293T with psPAX2 and pMD2G. Viral particles were collected 48 h and 72 h later, centrifuged them together at 4000 rpm for 5 min at 4 degree, and filtered with 0.45 μm filter. A549 cells were then infected with the viral particles, transfected with pLenti-miR-199a, or pLenti, were sorted for green fluorescence via flow cytometry.