Hct116

HT-29 (ACC 299) and HCT-116 (ACC 581) cells (DSMZ, Braunschweig, Germany) were maintained in McCoy’s5A and DMEM medium, respectively. U-2 OS cells (ATCC, Manassas, VA, USA) were kept in DMEM. All cell lines were tested for mycoplasma contamination on a regular basis. Media received 10% FBS, 1% sodium pyruvate and 1% penicillin/streptomycin concentrate. For 5-LO KO, cells were transiently transfected with the plasmid ‘lentiCRISPRv2’ (#52961, Addgene, Watertown, MA, USA) carrying a gRNA directed either to exon 2 (target sequence TGGATCACCGGCGATGTCGagg; HCT-116 F5, G6, H11; HT-29 F4; U-2 OS C4) or exon 6 (target sequence TGCAGCGCCGGATCAACACagg; HT-29 A2, G6; U-2 OS C4, H5) of the ALOX5 gene using Lipofectamine^TM LTX Reagent with PLUS^TM Reagent (Thermo Fisher^TM). After 72 h cells underwent puromycin selection and limiting dilution. Control cell lines received the empty ‘lentiCRISPRv2’ plasmid and were used as mixed clones.

The human breast cancer cell lines MCF-7, SK-BR-3 and MBA-MB-231 (triple-negative), LNCap (human prostate carcinoma lymph node metastasis), HeLa (cervix carcinoma), HepG2 (hepatocellular carcinoma), and HCT116 (human colon carcinoma) were purchased commercially from DSMZ (Braunschweig, Germany). If not otherwise stated, all cell culture media and supplements were obtained from Life Technologies (Darmstadt, Germany).
Cells were cultured as described previously [36 (link)]. In detail, MCF-7 in RPMI medium containing 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 0.1 M nonessential amino acids (NEAA); HeLa and HepG2 in RPMI medium containing 10% FBS and 2 mM Glutamax; MDA-MB-231 in Leibovitz’s L15 with 10% FBS; HCT116 and SK-BR-3 in McCoy’s 5A medium containing 10% FBS and 2 mM Glutamax; and LNCap in RPMI medium containing 10% FBS, 1 mM sodium pyruvate, 0.1 M NEAA, 2 mM Glutamax, and 0.01 M HEPES. All cell culture media were used without antibiotics. MCF-7, HCT116, SK-BR-3, LNCap, HeLa, and HepG2 were maintained at 37 °C in a humidified incubator with a 5% CO₂ atmosphere and MDA-MB-231 cells were handled without gaseous exchange. The absence of mycoplasma in the cell culture was regularly tested using the Venor GeM qOneStep-Kit (Minerva-biolabs, Berlin, Germany).

Human colorectal carcinoma cell line HCT-116 (LGC Standards, Wesel, Germany), Swiss albino mouse macrophage cell line J774, and fibroblast cell line 3T3 (both from DSMZ, Braunschweig, Germany) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany) in a humidified environment at 37 °C with 5% CO₂.
For SERS experiments, cells were grown on glass cover slips for 24 h prior to nanostar incubation. Then, a dilution of gold nanostars in DMEM-FCS of 1:10 (nanostar concentration ~5 × 10⁻¹² M) was added and cells were incubated with the nanostars for different times. Prior to SERS measurements, the cells adhering to the glass cover slips were rinsed two times with phosphate-buffered saline (PBS, Biochrom, Berlin, Germany) to eliminate remaining culture medium and nanostars, and kept in PBS. For cryo soft X-ray nanotomography, cells were grown on Formvar-coated gold grids and incubated in a similar fashion as described for SERS experiments. After the stipulated incubation time, each grid was rinsed three times with PBS, the excess of buffer was removed with a filter paper, and the grids were plunge-frozen in liquid ethane.