Mycoalert kit

Human Ma-Mel-19 (ECACC 13012460) and Sk-Mel-28 (ATCC HTB-72), and Mouse B16-F10 (ATCC CRL-6475) melanoma cell lines were purchased from Sigma Aldrich and periodically tested with MycoAlert^TM kit (Lonza, Switzerland). Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin (Euroclone). Sk-Mel-28 luciferase Luc2 (Promega, Italy) stable transfection was obtained with Lipofectamine LTX (Thermo Fisher) and by selection with hygromycin B (0.2 mg/mL; Sigma-Aldrich). miR-495-3p, miR-6730-3p, miR-376c-3p, or scramble control (mirVana miRNA mimics, Ambion) were transfected with Lipofectamine RNAimax (Thermo Fisher).

A549 were cultured in fully supplemented Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 4 mM L-glutamine and 10% (m/v) fetal bovine serum (FBS). Cells were grown at 37 °C in a humidified atmosphere with 5% CO₂. Prior to experimental work, cells were confirmed to be mycoplasma negative using the MycoAlertTM Kit (Lonza, Basel, Switzerland).

SW620 (CLL-227), HCT116 (CLL-247), MDA-MB-468 (HTB-132) and MDA-MB-231 (HTB-26) cells were from ATCC. PA-TU-8988T cells were from DSMZ (ACC162). SW620 and HCT116 WT cells were authenticated by short-tandem repeat analysis. SW620 MTHFD2^−/− and SW620 SHTM1^−/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2^−/−)^{17 (link)} or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1^−/−). MDA-MB-468 MTHFD2^−/− cells were generated as described previously^{24 (link)}. All cell lines were cultured in RPMI GlutaMAX (Life Technologies), except for PA-TU-8988T cells that were maintained in DMEM GlutaMAX (Life Technologies). Unless otherwise specified, all media were supplemented with 10% heat-inactivated FBS (Life Technologies), 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin (Life Technologies). Cell lines were maintained at 37 °C in 5% CO₂ humidified incubators, and routinely tested for mycoplasma using MycoAlertTM Kit (Lonza). HPLM was supplemented with 5% dFBS (Thermo Fisher Scientific). For experiments, 5 or 10% heat-inactivated or dFBS was used as indicated.

S2 cells were cultured in an insect medium (Hyclone, Amersham, UK) supplemented with 10% FBS (fetal bovine serum, Hyclone) and Penicillin/Streptomycin (Invitrogen, Carlsbad, USA). The MycoAlert^TM kit (Lonza, Basel, Switzerland) was used for the mycoplasma detection and validation of the authenticity of S2 cells. For aspirin treatment in S2 cells, we first prepared a 5 and 10 mM aspirin solution (dissolved in sterile H₂O). Then, 0.1 mL of each solution was added into S2 cells (0.9 mL) to reach the final concentration of aspirin, this being 0.5 or 1 mM, respectively. An equal volume of sterile H₂O was added in the control group. Six hours after aspirin treatment, S2 cells were infected with DCV (MOI 1), CrPV (MOI 0.1), FHV (MOI 1), VSV (MOI 1), or IIV6 (MOI 1).

Raji, a human Burkitt lymphoma cell line, was purchased from the American Type Culture Collection (ATCC). Raji cells were cultured in RPMI (RPMI 1640; Lonza, Switzerland) supplemented with 10% foetal bovine serum (FBS, Life Technologies, USA), 100 U/ml penicillin and streptomycin (PS), and 2 mM L-glutamine, 1 mM sodium pyruvate (all from PAA, Germany), and 1 mM HEPES (Sigma-Aldrich, Germany). Panc-1, a human pancreatic cancer cell line, was purchased from the European collection of cell cultures (ECACC). A375, a human melanoma cell line, was previously described.^{13 (link)} Panc-1 and A375 cells were cultured in DMEM3 + (DMEM with 10% FBS, 100 U/ml PS, and 2 mM L-glutamine). The retroviral amphitropic packaging cell line Platinum A was purchased from Cell Biolabs (USA). DMEM medium for Platinum A cells additionally contained 10 µg/ml puromycin and 1 µg/ml blasticidin (both from Sigma-Aldrich, Germany). The cell lines used in experiments were regularly checked for mycoplasma with the MycoAlert^TM kit (Lonza, Basel, Switzerland). Primary human T cells were cultured in VLE-RPMI 1640 (Biochrom, Germany) supplemented with 2.5% human serum (Sigma-Aldrich, Germany), 100 U/ml PS and 2 mM L-glutamine, 1 mM sodium pyruvate, 10 µM NEAA (Sigma-Aldrich, Germany), and 50 µM β-mercaptoethanol (human TCM).

The EBV-positive, latently I-infected cell lines Akata and MUTU-1 and the EBV-negative cell line BJAB (all BL cell lines) were cultured in RPMI 1640 GlutaMAX (ThermoFisher, Waltham, MA), supplemented with 10% (vol/vol) fetal bovine serum (FBS; Atlanta Biologicals, GA) and 1% penicillin-streptomycin (ThermoFisher, Waltham, MA). All cell types used for the present studies had routine mycoplasma testing done by a Lonza Myco-Alert kit (LT37–618; Lonza, Walkersville, MD), as per manufacturer’s instructions, and only mycoplasma negative cells were used in these studies.