Eagle s mem

Human pancreatic cancer cell lines BxPC3, HPAF2, Panc1, human colon adenocarcinoma cell lines Caco2, LS174T, and human lung adenocarcinoma cell line A427, NCI-H292 were obtained from the American Type Culture Collection. HPAF2, LS174T, and Caco2 cells were cultured in Eagle's MEM (Sigma, MO, USA), PANC1 and A427 cells were cultured in DMEM (Sigma, MO, USA), and BxPC3 and NCI-H292 cells were cultured in RPMI 1640 (Sigma, MO, USA). The media was supplemented with 10% fetal bovine serum (Invitrogen, Tokyo, Japan) and 100 U/mL of penicillin and 100 μg/mL streptomycin (Sigma, MO, USA). Hypoxic culture conditions were achieved with a multi-gas incubator containing a gas mixture of 94% N₂, 5% CO₂ and 1% O₂ (ASTEC, Fukuoka, Japan).

1BR (human fibroblasts) hTERT cells or A549 cells were cultured in the Alpha modification of minimum essential medium (MEM) (Wako, Osaka, Japan) or Eagle's MEM with 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA), respectively. X-ray irradiation was performed using a Faxitron RX-650 (100 kVp, 1.14 Gy/min, Faxitron Bioptics, Tucson, AZ, USA) with a dose rate of approximately 1 Gy/min. C-ion irradiation was performed at Gunma University Heavy Ion Medical Center; GHMC (290 MeV/n, LET 20 or 60 keV/μm) [45 (link)]. The LET value at the irradiation position was derived using Monte Carlo simulations. The position of the Bragg peak in the physical dose distribution was measured using an ionisation chamber at depth in a water phantom. The depth in the water phantom was adjusted so that the cells were placed 2.2 mm before the Bragg peak to set up 60 keV/μm. To visualize the particle track, horizontal irradiations were conducted with a mono-energetic broad beam.

SW480 colon cancer cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Two SW480 knockout cell lines, SW480-KO16 and SW480-KO55, were generated at our laboratory using the predesigned RhoB-human gene knockout kit via CRISPR (#KN209837, OriGene Technologies, Rockville, MD) [14 (link)]. The three cell lines were maintained in Eagles MEM (Sigma-Aldrich, St. Louis, MO) with 10% heat-inactivated fetal bovine serum albumin (GIBCO, Invitrogen, Paisley, UK) and 2 mM L-Glutamine at 37 °C and 5% CO2 at that temperature (Life Technologies, Carlsbad, CA). HCT116 colon cancer cell line was received from the Johns Hopkins University core cell center and kept in McCoy's 5A medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum albumin (GIBCO, Invitrogen, Paisley, UK) at 37 °C and 5% CO2.

The MCF7 cell line was maintained in Eagle’s Minimum Essential Medium (Eagle’s MEM) (Sigma, M0268) and the MDA-MB-231 cell line in Dulbecco’s Modified Eagle’s Medium/Nutrient blend F-12 Ham (DMEM/F12) (Caisson, DFP18), both supplemented with 10% fetal bovine serum (Biowest, BIO-S1650), under 5% CO₂ atmosphere at 37°C.