Plvx ires mcherry

To overexpress the indicated TFs, the canonical CDS at each gene locus was obtained from NCBI GenBank. After necessary sequence optimization for gene cloning, DNA sequences were synthesized and sub-cloned into the lentivirus vector pLVX-IRES-mCherry (Takara, Tokyo, Japan). The TF overexpression vectors were co-transfected into 293 T (CRL-11268, American Type Culture Collection) cells with the 2^nd-generation lentivirus packaging plasmids psPAX2 and pMD2G, and the virus in the medium supernatant was harvested and concentrated for target cell infection.

The STR-verified HNSCC cell lines UM-SCC-1, UMSCC-17B, Detroit 562, PCI-13, MDA1586, PCI-15B, TR146, and Ca9-22 were described previously [46 (link)], and STAR cells was established in our laboratory. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. Stable cell lines expressing control vector pBabe; wild-type p53; mutant p53s G245D, C238F, R175H, and E336X; and short hairpin RNAs (shRNAs) for p53 (shp53) and AMPKα1 (shAMPK α1) were established as described previously [9 (link), 16 (link)]. pBMN-GFP (Addgene, Cambridge, MA, USA), pLVX-IRES-mCherry (Takara Bio USA, Mountain View, CA, USA), and the PCR product from pcDNA3-FLAG-FKHRL1 (Addgene) were used to generate retroviral pBMN-GFP-FOXO3a and lentiviral pLVX-IRES-mCherry-FOXO3a expression. Lentiviral pGIPz shRNA constructs shFOXO3A-A (V3LHS_375381), shFOXO3A-B (V3LHS_375386), shFOXM1-A (V2LHS_283849), shFOXM1-C (V3LHS_396939), shp53-A (V3LHS_333920), and shp53-C (V3LHS_333919) were from Dharmacon (Lafayette, CO, USA). AMPKα1 and FOXO3a CRISPR/Cas9 knockout (KO) plasmids sc-400104 and sc-400308 were from Santa Cruz Technologies (Dallas, TX, USA).

Full‐length wild‐type ECE1c cDNA with in‐frame 5′‐Flag and Myc tags was inserted into the multicloning region of plasmid pLVX‐IRES‐mCherry (Clontech, Mountain View, CA, USA). Site‐directed mutagenesis was performed using the GENEART kit (Invitrogen) according to manufacturer’s instructions. Lenti‐X 293T cells were transfected with 8 μg psPax2 (encoding Gag‐Pol protein), 8 μg pLVX‐IRES‐mCherry (encoding ECE1c^WT, ECE1c^K6R or empty), and 4 μg pCMV‐VSVg (encoding VSV G‐glycoprotein) and then suspended in 500 μL 0.25 m CaCl₂. At 48 h post‐transfection, supernatants containing pseudotyped particles were harvested and passed through a cellulose acetate filter with a pore size of 0.45 μm. Viral particles were purified and concentrated by ultracentrifugation at 150 000 g for 75 min in SureSpin 630 rotor (Thermo Fisher, Vilnius, Lithuania) through a 25% sucrose cushion (TNE‐Sucrose 25%). Finally, cells were cultured at 5 × 10⁴ cells/well in 12‐well plates along with the recombinant lentiviruses at a MOI of 5 under normal growth conditions. Expression of mCherry was examined 72 h post‐transduction under a Nikon Eclipse TS100 Inverted Microscope (Nikon, Tokyo, Japan) equipped with epifluorescence. Cells were expanded for 1 week, and the brightest (mCherry⁺) cells were sorted on a FACSAria Fusion cell sorter (Becton‐Dickinson, San Jose, CA, USA).

To detect eMSC in vivo, the cells were transduced with a mCherry lentivirus. Lentivirus was generated by co-transfection of three plasmids; 10 µg pLVX-IRES-mCherry (lentivirus plasmid which contains mCherry gene) (clontech-6312237), packaging plasmids; 9 µg pSPAX2 (which encodes capsid) (Addgene 12260) and 1 µg pMD2.G (encodes reverse transcriptase for lentivirus replication) (Addgene 12259), into 293 cells. (https://www.addgene.org/protocols/bacterial-transformation/) using the TransIT-X2 (Mirus) transfection reagent according to manufacturer’s protocols. Transfection was confirmed by monitoring mCherry expression by fluorescence microscopy. Viral containing supernatant was collected and passed through a 0.45um filter. eMSCs were grown to 70% confluence and transduced with lentiviral supernatant supplemented with polybrene (5ug/ml) (Sigma hexadimethrine bromide, catalogue #107689). Viral supernatant was replenished after 6 hours.