Acl 4

All lung cell lines were obtained directly from the American Type Culture Collection (ATCC, Manassas, VA, USA). Lung SCC lines H226, H2170, H520, H596, and ChaGo‐k‐1 were maintained in RMPI‐1640 (Nacalai Tesque), Calu‐1 in McCoy's 5a (Nacalai Tesque, Kyoto, Japan), SK‐MES‐1 in DMEM (Nacalai Tesque), SW900 in Leibovitz’s medium (Gibco, Life Technologies, Carlsbad, CA, USA), H1869 in ACL‐4 (Gibco, Life Technologies), and H2066 in HITES medium (Gibco, Life Technologies). Normal lung fibroblast cells (MRC‐5, IMR‐90, and WI‐38) were cultured in EMEM (Gibco, Life Technologies). All media were supplemented with 10% fetal bovine serum, 2 mm l‐glutamine, 100 μg·mL⁻¹ streptomycin, and 100 U·mL⁻¹ penicillin. ACL‐4 and HITES were further supplemented with additional nutrients as recommended by the ATCC. All cell lines were authenticated with GenePrint^® 10 System (Promega, Fitchburg, WI, USA).
For inhibitor studies, belinostat (PXD101), bortezomib (Velcade), GDC0879, PD0325901, RDEA119, and GSK1120212 were obtained from Selleck Chemicals (Houston, TX, USA); cisplatin from Hospira (Lake Forest, IL, USA); MG‐132 from Sigma (St. Louis, MO, USA); and cetuximab was from Merck (Darmstadt, Germany).

NCI-H1975, Ramos, and SNU-16 cells were maintained in Revolutions-Per-minute Indicator 1640 (RPMI 1640) medium (Gibco, Grand Island, NY, USA), A431 was maintained in Dulbecco’s Modified Eagle Medium (DMEM) medium (Gibco), MDA-MB-231 was cultured in L-15 medium (Gibco), and NCI-H1581 was cultured in ACL-4 (Gibco), and all of them were supplemented with 10% heat-inactivated fetal calf serum (FBS; Gibco) at 37 °C in a 5% CO₂ humidified environment. Cells in 96-well plates were treated with gradient concentrations of compounds at 37 °C for 72 h. Cell proliferation assays in NCI-H1975, A431, MDA-MB-231 and NCI-H1581 were determined by using sulforhodamine B (SRB; Sigma, St. Louis, MO, USA). The antiproliferative activity of compounds, examined in Ramos and SUN-16 cell lines, was determined by using CCK-8 (Dojindo Laboratories, Kamimashiki-gun, Japan). The dosages corresponding to the half-maximal inhibition (IC₅₀) were calculated using SoftMax pro-based nonlinear 4-parameter regression analysis (n = 3).

A total of 1×10⁶ cells were washed twice with PBS and then subjected to subcutaneous flank injection into NOD.SCID mice (The Jackson Laboratory, Jacksonville, FL). Freshly prepared human ACC tumor obtained under IRB-approved tissue protocol was also subjected to xenograft tumor growth and short term cell culture. Xenograft tumor volumes were calculated as: volume = (width)² × length/2. The primary tumor cell culture was performed using methods as described in [43 (link), 76 ]. The media used for primary tumor cell culture includes F12K (Sigma) 1:1 mixture with DMEM (Sigma) supplemented with 5-20% FBS (Gibco) and ACL4 medium supplemented with 5% FBS.