Plvx ires zsgreen

HEK293T cells stably expressing human ACE2 and TMPRSS2 were generated by lentiviral transductions as previously described^{23 (link)}. Briefly, the open reading frames for hACE2 (Addgene, 1786) and hTMPRSS2a (IDT, synthetic gene fragment) were cloned into lentiviral expression vectors pRRLsinPPT.CMV.GFP.WPRE^{52 (link)} and pLVX-IRES-ZsGreen (Clontech, 632187), respectively. For ACE2 cloning, Age1/Bsrg1 cut sites were used to replace GFP with ACE2, while hTMPRSS2a was cloned into pLVX-IRES-ZsGreen using EcoR1/XhoI restriction sites. Lentiviral particles expressing the above genes were produced by co-transfecting expression plasmids individually with a 2nd generation lentiviral packaging construct psPAX2 (courtesy of Dr Didier Trono through NIH AIDS repository) and VSVG plasmid pMD2.G (Addgene, 2259) in HEK293T producer cells using polyethyleneimine as previously described^{53 (link)}. Virus supernatant was collected 72 h post-transfection, pre-cleared of cellular debris and centrifuged at 28,000 × g for 90 min at 4 °C to generate concentrated virus stocks. Two successive rounds of lentiviral transductions were then performed on HEK293T cells to generate ACE2-TMPRSS2 HEK293T cells. Clonal selection led to the identification of a highly permissive clone (HAT-24), which was then used in subsequent experiments^{23 (link)}.

Overexpression of Dlk1^M in Dlk1⁻ cells was achieved with lentivirus based on a pLVX-IRES-zsGreen (Clontech, Japan) vector containing the Dlk1^M sequence amplified from mouse cDNA with the forward primer 5′-CCGGAATTCATGATCGCGACCGGAGCCCT-3′ and reverse primer 5′-CGCGGATCCTTAGATCTCCTCATCACCA-3′. 293T cells were transfected with mock or Dlk1^M vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Lentivirus was collected 3 days later and used to transduce Dlk1⁻ cells using Lipofectamine 2000 as previously described [21 (link)].

HPV-18 E2 ORF was synthetized and cloned by Genscript into the bicistronic lentiviral vector pLVX-IRES-ZsGreen (Clontech, Mountain View, CA, USA). The sequence was partially modified to optimize translation of E2 (Supplementary Fig. 1). Lentiviral particles were produced by co-transfection of lentiviral vector encoding HPV-18 E2, or the empty vector as control (ZsGreen), with the ViraPower packaging system (Invitrogen, MA, USA) into HEK293T cells, using Lipofectamine2000 (Invitrogen, MA, USA) according to manufacturer’s directions [14 (link)]. After 48 h, viral particles were harvested and concentrated by ultracentrifugation, and the viral titer was determined by serial dilutions using HEK293FT cells, followed by flow cytometry analysis of ZsGreen expression 72 h post-transduction (BD FacsCanto II).

To validate the candidate target genes predicted by bioinformatics analysis are targets of miR-630, 3′UTRs of ARFGEF2, PDGFRA, SET, MTDH and EP300 containing the miR-630 binding site were amplified from the human genomic DNA using primers ARFGEF2-UTR-F/R, PDGFRA-UTR-F/R, SET-UTR-F/R, MTDH-UTR-F/R, severally (Supplementary Table S1), and then were cloned into the downstream of Renilla luciferase gene in the psiCHECK™-2 vector (Promega). Mutant MTDH-UTR plasmid containing three mutated bases on the predicted binding site was constructed using the fast mutagenesis kit (Vazyme) with primers MTDH-UTR-mut-F/R.
To stably overexpress mature miR-630 in MDA-231-D3H2LN cells, the DNA fraction containing the mature sequence of miR-630 was amplified with primers miR-630-F/R (Supplementary Table S1) from the model plasmid pcDNA^TM6.2-GWSW/miR-630 kindly provided by Prof, Min-Liang Kuo (National Taiwan University) and then was cloned into the lentiviral expression plasmid pLVX-IRESZsGreen (Clontech Laboratories). The pcDNA3.1-MTDH plasmid was structured previously in our lab [25 (link)].
For co-expression of microRNA mimics and its target-genes, the target-gene MTDH was transfected using Hily Max (Dojindo, Kumamoto, Japan) firstly, and after 24 hours the microRNA mimics were transfected using Lipofectamine 2000 reagent (Invitrogen).

To confirm the possibility of miR-494 targeting predicted candidate gene, 3′UTR of APC, Rab5A and PAK1 containing miR-494 binding site were cloned into the downstream of Renilla luciferase gene in the psiCHECK-2 vector (Promega). Mutant PAK1-3′UTR containing single mutated base and double mutated base sites were constructed using fast mutagenesis kit (Vazyme, shanghai, China). For PAK1 overexpressing, PAK1 was cloned into the pcDNA3.1. And pri-494 was cloned into the lentiviral expression plasmid pLVX-IRES-ZSGreen (Clontech Laboratories, CA, USA) for miR-494 stable overexpressing.