Normocin

SUM159 cells were propagated in Nutrient Mixture F-12 supplemented with 5% fetal calf serum, 0.5 μg/ml hydrocortisone, and 10 μg/ml insulin (all from Sigma), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml Normocin (InvivoGen). MCF10A-HER2/HER3 [1 (link)] were propagated in DMEM/F12 medium (Invitrogen) supplemented with 5% horse serum (Hyclone), 20 ng/ml human EGF (Peprotech), 0.5 μg/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 μg/ml insulin (all from Sigma), 100 IU/ml penicillin, 100 μg/ml streptomycin and 100 μg/ml Normocin (InvivoGen). These cells were profiled for cell line-specific highly-polymorphic short tandem repeat loci (STRs) (Microsynth). Previously published tools were used for the inducible RNAi studies [1 (link)]. For siRNA experiments, 175,000 cells were seeded in 6-well plates. The following day, cells were transfected using DharmaFECT and the human ETS1 siRNA- SMARTpool (L-003887-00-0005) or the non-targeting pool as control (D-001810-10-05). Experiments were performed according to the manufacturer’s protocol and using siRNA at final concentrations of 25 nM and 12.5 nM for SUM159 and MCF10A-HER2/HER3, respectively. Cells were harvested at the times indicated in the figure legends.

Human multiple myeloma cell lines L363, KMS12PE, JIM1, AMO1, KMM1, KMS27, ARD, OPM1, KHM11, KARPAS417, and H929 were cultured in Advanced RPMI 1640 (6% heat-inactivated fetal bovine serum (FBS): Gibco by Life Technologies, 2 mM l-glutamine: Gibco by Life Technologies, 100 U/mL Penicillin and 100 μg/mL Streptomycin: Gibco by Life Technologies, 100 µg/mL Normocin: InvivoGen) and incubated at 37 °C with 5% CO₂. Human Burkitt′s lymphoma cell line CA46 was cultured in RPMI 1640 (10% heat-inactivated fetal bovine serum (FBS): Gibco by Life Technologies, 2 mM l-glutamine: Gibco by Life Technologies, 100 U/mL Penicillin and 100 μg/mL Streptomycin: Gibco by Life Technologies, 100 µg/mL Normocin: InvivoGen) and incubated at 37 °C with 5% CO₂. Media was changed every 2 days. For cells plated and harvested for protein or RNA, pellets were washed twice with cold PBS. After aspirating off the PBS, pellets were flash frozen in dry ice and transferred to a −80 °C freezer for short term storage. Cell lines were obtained from Michael Kuehl (NCI) and tested by CNV fingerprinting to verify their authenticity⁶⁴ .

Murine monocyte macrophage Raw 264.7 cells, human colon carcinoma T84 cells and Caco2 were cultured in T75 flasks in Dulbecco’s Modified Eagles Medium (MEM, 1:1 with sodium bicarbonate (Life Technologies, USA)). DMEM with sodium bicarbonate was used for growth of HT29. Each media was substituted with L-glutamine, 10% heat inactivated fetal bovine serum and 100 μg/ml Normocin (Invivogen, USA). Human primary alveolar epithelial NuLi-1 cells were cultured similarly in Bronchial Epithelial Growth Medium (BEGM) supplemented with 100 μg/ml Normocin (Invivogen, USA) without FBS. Each cell line was cultured under 5% CO₂ in a water-jacketed CO₂ incubator until they were confluent. For the ELISA tests, approximately 4 × 10⁴ cells were seeded in a well of regular 12 well tissue culture plates (SPL Life Sciences, South Korea). T84 intestinal epithelial cells were seeded onto 12 mm Costar^® polystyrene (Corning Inc. USA) trans-well plates (0.3 μm pore size, 0.33 cm² insert diameter, 10⁶ cells/cm² and allowed to grow for two weeks to obtain polarized epithelial cells. The trans-well inserts were equilibrated in culture medium for at least 2 hours before seeding. The media in each plate was changed as needed.

THP1, Jurkat, Hela, L929 and RAW264.7 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). THP1-Lucia ISG cells were purchased from Invivogen. cGAS^-/- Hela cells were a kind gift from Dr. Zhengfan Jiang (School of Life Science, Peking University, Beijing, China). Human peripheral blood mononuclear cells (PBMCs) were purchased from LDEBIO (Guangzhou, China). THP1, THP1-Lucia ISG cells, Jurkat and PBMCs were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gbico) and 0.1% Normocin (InvivoGen, Cat# ant-nr) and 0.1% mycoplasma elimination reagent (Yeasen, Cat# 40607ES03) at 37 °C with 5% CO₂. Hela, cGAS^-/- Hela, L929 and RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gbico) supplemented with 10% FBS and 0.1% Normocin (InvivoGen, Cat# ant-nr) and 0.1% mycoplasma elimination reagent (Yeasen, Cat# 40607ES03) at 37 °C with 5% CO₂.

EV-G was isolated from intestinal homogenate submitted to Newport Laboratories in August, 2013, from commercial 4-week old pig with diarrhea for diagnostic testing.
Rhesus monkey kidney cells (Marc-145) (ATCC CRL-12219) were used for virus isolation. The Marc-145 cells were propagated in DMEM (Pellgro, Manassas, VA) with either plasmocin (25 mg/l) (Invivogen, San Diego, CA) or normocin (100 mg/l) (Invivogen). Confluent cells were washed 2–3 times with replacement media (DMEM (Pellgro) with trypsin (EDTA) at 5 µg/mL and either plasmocin (25 mg/l) (Invivogen, San Diego, CA) or normocin (100 mg/l) (Invivogen). The diagnostic sample, intestinal homogenate, was centrifuged at 5000×g for 10 min to remove debris. Supernatant was used to inoculate confluent, washed Marc-145 cells. The cultures were incubated at 37°C in a CO₂ incubator and inspected daily for cytopathic effect (CPE). If CPE was noted, media was collected and analyzed for common enteric viruses (rotaviruses A, B, and C) by quantitative reverse-transcription polymerase chain reaction (RT-PCR) (data not shown).

THP-1 cells were obtained from Japanese Collection of Research Bioresources Cell Bank (Osaka) and grown in an RPMI medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10 % (vol/vol) fetal bovine serum (Thermo Fisher Scientific), 1 % (vol/vol) GlutaMAX™ Supplement (Thermo Fisher Scientific), and 100 μg/ml Normocin™ (InvivoGen, Sandiego, CA) at 37° C with 5 % CO₂. THP1-Xblue™-MD2-CD14 cells were acquired from InvivoGen and grown in an RPMI medium supplemented with 10 % fetal bovine serum, 100 μg/ml Normocin™ (InvivoGen), 200 μg/ml Zeocin™ (InvivoGen) and 250 μg/ml G418 (InvivoGen) at 37° C with 5 % CO₂ . THP1-Xblue™-MD2-CD14 cells contain the secreted embryonic alkaline phosphatase (SEAP) reporter gene under controls of NF-κB and/or activator protein-1 (AP-1).