Lipofectamine 3000 transfection reagent

For overexpression experiments, YAP–HaloTag CRISPR knock-in U-2 OS cells were transfected with pEGFP-C3-Lats1 (Addgene plasmid # 19053) or pEGFP C3-Mst2 (Addgene plasmid # 19056), both gifts from Marius Sudol, pRK5-Myc-Fus-R495X,⁶⁸ (link) gift from Jiou Wang, or EGFP-TAZ (made in the Cai lab) using Lipofectamine 3000 Transfection Reagent (cat. no. L3000015), for 16 h. For RNAi experiments, a mixture of Lats1 siRNA (Thermo Fisher, Silencer Select s17393) and Lats2 siRNA (Thermo Fisher, Silencer Select s25503) was used at a final concentration of 10 nM, or the scrambled negative control siRNA was used at a final concentration of 20 nM (Thermo Fisher, AM4611), and were transfected into cells using the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher, cat. no. 13778075) for 48 h. For siTEAD1 experiments, the TEAD1 siRNA (Thermo Fisher, Silencer Select s13962) or scrambled negative control siRNA was used at a final concentration of 10 nM (Thermo Fisher, AM4611), and were transfected into cells using the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher, cat. no. 13778075) for 48 h, after which cells were replated for live cell imaging and RT-qPCR.

The Flp-In™ T-REx™ HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM GlutaMAX-I, 10% FBS, and 100 U/mL HyClone penicillin-streptomycin at 37 °C and 5% CO₂.
The pcDNA5-GFP-Vimetin construct was generated by inserting vimentin cDNA (NCBI Reference Sequence: NM_003380.5) into the pcDNA5-GFP backbone. Plasmid mCherry-LaminA-C-18 (#55068) was ordered at ADDGENE.
The cells were seeded at 1 to 2 × 10⁵ per 6-well, and plasmid transfections were performed using Lipofectamine™ 3,000 transfection reagent. The fluorescent-positive cells were sorted with FACS into a 6-well plate. The positive cells were grown in a selection medium with G418 (200 μg/mL), hygromycin (100 μg/mL), and blasticidin (15 μg/mL). The expression of the vimentin protein was induced with tetracycline at 1 μg/mL for 24 h.