Lipofectamine 3000

The human chordoma cell lines U-CH1 and U-CH2 were both obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in a 1:4 ratio of Iscove’s modified Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) and RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco) in a humidified incubator with a 5% CO₂/95% air atmosphere at 37 °C. Culture flasks were coated with rat tail type I collagen (BD Biosciences, San Diego, CA, USA) prior to use. The following antibodies were used in the experiments: anti-Vimentin, anti-N-cadherin and anti-GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA), and anti-Smad3 and anti-E-cadherin were obtained from Abcam (USA). Smad3 small interfering RNA (siRNA) was purchased from Suzhou GenePharma (Suzhou, China). Lipofectamine 3000 was purchased from Origene (Rockville, MD, USA).

The CDKN1A (encoding p21^waf1) promoter-eGFP reporter plasmid (pEZX-PF02) (HPRM51895-PF02, GeneCopoeia) was transfected in early-passage IMR-90 cells (PN16) using Lipofectamine 3000 (ThermoFisher) according to the manufacturer instructions. Forty-eight hours post-transfection, cells were cultured with 2 µg.ml⁻¹ puromycin for >2 weeks to select stable integrations. The resultant stable cell population expressing GFP under the control of the CDKN1A promoter was then either not transduced or transduced with shSCR or shCSB#2, as described in the previous paragraph.
Ectopic expression of CSB was obtained upon transfection with Lipofectamine 3000 of early-passage IMR-90 (PN15) with the CSB (Myc-DDK-tagged) expression vector (pCMV6-Entry) (RC219020, OriGene), called here “pCSB”. Transfection with pcDNA3.1 (ThermoFisher Scientific, V79020), which codes for neomycin resistance under control of a CMV promoter or “Empty vector” was used as control. The CSB coding sequence was verified by sequencing pCSB before transfection. pCSB was amplified in Stbl2^TME.coli (ThermoScientific) at 30 °C, since amplification of pCSB in classic DH5 α E.coli resulted in multiple mutations (as indicated by the supplier). Transfected cells were kept in culture in the absence of selection for 8 days, including a passage in culture, before analysis.

Lung cancer cells and B16F10 cells were transfected with negative control (NC) or ApoE siRNA (Santa Cruz Bio) using Lipofectamine® RNAiMAX reagent (Invitrogen) in Opti-MEM, following the manufacturer's protocol. For ApoE stable knockdown cells, B16F10 cells were transfected with NC or ApoE shRNA (pGFP-V-RS vector; Origene) using Lipofectamine® 3000. Transfected cells were cultured with Puromycin (Sigma-Aldrich) for 3 weeks. Puromycin-resistant colonies were selected and expanded.

Gene knockdowns in RAW 264.7 cells were performed by transfecting cells with siRNA. Cells were plated at a density of 5 × 10⁶ per well in six-well plates and allowed to attach for 20 h. Cells were transfected with 100 nM siRNA against FAK using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 h, cells were treated as indicated. To express FAK in macrophages, a recombinant plasmid vector coding FAK cDNA was obtained from Sino Biological (Cat No.: MG50470-NF). To inactivate FAK by site-directed mutation, the FAK Tyr397 to Phe397 variant (Y397F) obtained from TSINGKE Biological (Nangjing, China) was used. These plasmids were transfected into RAW 264.7 cells using Lipofectamine 3000.
To overexpress FAK, FAK mutation, and TAK1 in tool cells, three plasmids respectively encoding Flag-labeled wide-type FAK (FAK-WT-Flag), Flag-labeled FAK Y397F mutation (FAK-Y397F-Flag), and Myc-labeled TAK1 (TAK1-Myc) were designed and obtained from OriGene Technologies (Beijing, China). These three plasmids were transfected into 3T3 cells using Lipofectamine 3000.