Ptripz vector

Human wild type (Wt), N-terminal myristoylated (myr)-tagged, and C-terminal NLS-tagged PTEN complementary DNA (cDNA) sequences were subcloned from pCDNA3-PTEN into pTRIPZ vector (Addgene, Watertown, MA, USA) with AgeI and MluI to generate PTEN expression plasmid. All plasmid constructs were confirmed by sequencing. Ampicillin, puromycin, and doxycycline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyethylenimine (PEI) was purchased from Polysciences (Warrington, PA, USA), while RPMI, DMEM, Opti-MEM reduced serum media, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and Protein G Sepharose 4 Fast Flow beads were purchased from Cytiva (Malborough, MA, USA). The antibodies for Western blotting were as follows: anti-PTEN (138G6, 1:1000), anti-p-AKT (9271, 1:1000), anti-AKT (9272, 1:1000), anti-GAPDH (D16H11, 1:4000), anti-Lamin B1 (D4Q4Z, 1:1000), anti-β-Tubulin (9F3, 1:1000), and anti-EGFR (D3871, 1:1000) antibodies, all purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-HSP90 (610418, 1:4000) was purchased from BD Bioscience (Franklin Lakes, NJ, USA).

The open reading frames of mouse Tbx5, Tbx3, Tbx18, Shox2, or TAk1 were amplified by PCR and directionally cloned downstream of the TurboRFP reporter using ClaI and MluI restriction sites in the pTripZ vector (Addgene) viral constructs were then validated by sequencing. Lentiviruses were produced using the second-generation lentiviral expression system (Bajpai and Terskikh, 2007 ). Plasmids will be made available upon request to the corresponding author.