Wm115

Two human melanoma cell lines WM115 and WM266-4 were obtained from Rockland Immunochemicals (Limerick, PA, USA). Cells were cultured in RPMI 1640 Media (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (EURx, Gdansk, Poland) and 100 units/mL of Penicillin-Streptomycin mix (ThermoFisher Scientific, Waltham, MA, USA). Cells were cultured in normoxic (21% O₂) and hypoxic (1% O₂) conditions. Reduced oxygen culture conditions were obtained, as described previously [46 (link)]. All the cells were tested negative for mycoplasma using MycoCheckTM Mycoplasma PCR Detection Kit (MoBiTec Molecular Biotechnology, Goettingen, Germany).

The following cell lines were from the American Tissue Type Collection: A375, HEMN (normal adult epidermal melanocytes) and Jurkat. The following cell lines were from Rockland Immunochemicals: WM983A, WM983B, WM1366, WM115, WM266-4, WM164, WM793 and Lu1205. SW1 melanoma cells (gift from the Ronai laboratory at the Sanford Burnham Prebys Medical Discovery Institute) and SM1 melanoma cells (gift from the Smalley laboratory at the Moffitt Cancer Center) were cultured in DMEM containing 10% FBS, 1 g ml⁻¹ glucose and 4 mM l-glutamine at 37 °C with 5% CO₂. HEMN cells were grown in Lonza MGM-4 growth medium; before collection for IB analysis, the cells were switched to the same medium as the other cells overnight. Cell lines were transfected using Lipofectamine 2000 (Invitrogen). Primary CD4⁺ T cells were collected using the EasySep Human CD4⁺ negative selection isolation kit (Stemcell Technologies) according to the manufacturer’s protocols. Upon arrival at the laboratory, all cell lines are quarantined until they have passed footprint identification and mycoplasma testing (as mycoplasma negative). The identities of all cell lines (human and mouse) in the Lau laboratory are verified annually by short tandem repeat-based authentication ‘CellCheck’ services provided through IDEXX BioResearch.

B16F10, HeLa, A375, and RPMI7951 cells were purchased from American Type Culture Collection. WM115 and WM266-4 cells were purchased from Rockland Immunochemicals. HEK 293FT cells were purchased from Life Technologies. Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. HeLa cells were transfected with GFP, GFP-FAK, GFP-FERM, GFP-kinase, or GFP-FRNK constructs using polyethylenimine (Polysciences) (31 (link)). B16F10 cells were transduced with adenovirus encoding for either FAK-WT, FAK-KD, FAK FERM-WT, or FAK FERM-NLM.