Penicillin

A549 (CCL-185)- collected from a 58-year old Caucasian male, it is a hypotriploid human cell line with the modal chromosome number 66 which can be found in 24% of cells, NCI-H1299 (CRL-5803)- established from a lymph node of 43-year old White male patient with lung cancer who received prior radiation therapy, MDA-MB-231(HTB-26)- obtained from a 51-year old White female, it is an aneuploid female with a modal chromosome number 64, and MDA-MB-468 (HTB-132)- isolated from a pleural effusion of a 51-year old Black woman with a metastatic breast adenocarcinoma, an aneuploid female with most chromosome counts in the hypertriploid range with a modal chromosome number 64. The A549, MDA-MB-231, and MDA-MB-468 cells were cultured in high glucose Modified Eagle Medium (DMEM) (Genesee Scientific, San Diego, CA, USA) while NCI-H1299 cells were cultured in RPMI 1640 (Genesee Scientific, San Diego, CA, USA). All media were supplemented with 10% heat-inactivated fetal bovine serum (Genesee Scientific, San Diego, CA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Genesee Scientific, San Diego, CA, USA). The cultures were incubated at 37ºC in 5% CO₂/95% humidified air. In all cases, treatment with experimental compounds was done in basal medium supplemented with 5% heat-inactivated fetal bovine serum.

Three different firefly luciferase-expressing mouse cancer cell lines were used: 4T1-Luc2 (mammary carcinoma), B16F10-Luc2 (cutaneous melanoma), and MOC2-Luc2 (oral squamous cell carcinoma). The 4T1-Luc2 cells were cultured in Dulbecco’s modified eagle medium (DMEM, high in glucose with sodium pyruvate and glutamine, Fisher Scientific) with 10% fetal bovine serum (Genesee Scientific) and 500 µg/mL of zeocin (InvivoGen). The B16F10-Luc2 cells cultured in RPMI 1640 (Gibco) containing 10% fetal bovine serum (Genesee Scientific). The media was supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin (Genesee Scientific). The MOC2-Luc2 cells were cultured in HyClone™ Iscove’s Modified Dulbecco’s Medium (IMDM) and Hams F12 Nutrient Mixture (Fisher Scientific) at a 2:1 mixture with 5% fetal bovine serum, 1% penicillin/streptomycin, 5 ng/mL epidermal growth factor, 400 ng/mL hydrocortisone, and 5 mg/mL insulin (MilliporeSigma). All cells were incubated at 37°C in 5% CO₂ atmosphere and passaged 1:10 when 75-90% confluent. Cell lines underwent authentication via short tandem repeat analysis and PCR-based interspecies contamination testing performed by a third-party purveyor (CellCheck Mouse 19; IDEXX), and also underwent routine Mycoplasma testing.

All cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). MDA-MB-231, A549, and MIAPaCa-2 cells were cultured in high glucose Modified Eagle Medium (DMEM, Genesee Scientific, San Diego, CA, USA). NCI-H1299 cells were cultured in RPMI 1640 (Genesee Scientific, San Diego, CA, USA). All media were supplemented with 10% heat-inactivated fetal bovine serum (Genesee Scientific, San Diego, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Genesee Scientific, San Diego, CA, USA). The cultures were incubated at 37 °C in 5% CO₂/95% humidified air. In all cases, treatment with experimental agents was done in basal medium supplemented with 5% heat-inactivated fetal bovine serum. Antibodies specific to B-Raf (Cat. #14814), Phospho-B-Raf (Ser445) (Cat. #2696), c-Raf (Cat. #53745), Phospho-c-Raf (Ser338) (Cat. #9427), MEK1/2 (Cat. #8727), Phospho-MEK1/2 (Ser217/221) (Cat. #9154), p44/42 MAPK (Erk1/2) (Cat. #4695), RSK1/RSK2/RSK3 (Cat. #9355), Phospho-p90RSK (Ser380) (Cat. #11989), GAPDH (HRP Conjugate) (Cat. #8884), α-Actinin (HRP Conjugate) (Cat. #12413), and anti-rabbit IgG, HRP-linked Antibody (Cat. #7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).