Antibiotic antimycotic 100

Dental pulp stem cells (DPSCs, a kind gift from Dr. Shi, University of Pennsylvania) were cultured in growth media [α-minimum Eagle's medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% Antibiotic–Antimycotic (100 ×) (Thermo Fisher Scientific)] in a humidified CO₂ incubator at 37 °C. Cells were split at 1:3 ratio at ~ 90% confluency (passage), and cells from passage 6 to 12 were used in all experiments.

The intestinal tissue was opened longitudinally and cut into 0.5 cm small pieces, and kept in complete medium (RPMI‐1640; Lonza, Belgium) supplemented with 10% FBS (Thermo Fisher Scientific, Belgium) and 1% antibiotic–antimycotic 100× (Thermo Fisher Scientific), incubated at 37°C for 20 min, shaken vigorously for 15 s, and filtered through a 70 µm cell strainer (Greiner Bio-One, Belgium). Repeat the above operation and combine the filtrates collected twice. The remaining tissue was digested in RPMI-1640, which was supplemented with 1 mg/ml collagenase type I (Sigma, USA), 60 U/ml DNase I (Invitrogen) and 10% FCS, 220 rpm, 37°C for 60 min, and then filtered through a 70 µm cell strainer. The filtrate was centrifuged at 1,500 rpm for 5 min to collect the cell pellet and blocked in 5% BSA for 1 h for subsequent staining. Cell types were defined based on the following markers: macrophages (CD11b⁺ F4/80⁺), CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD8⁺), neutrophils (CD11b⁺Ly6G⁺) and ILC cells (CD127⁺). All the antibodies for FACS were purchased from BD Pharmingen (San Diego, CA). Fluorescence was measured with a flow cytometer (FACS Calibur; BD Biosciences) equipped with Cell Quest software (BD Biosciences, Canada).

The following human melanoma cell lines were used: WM3918, 501-Mel, Sk-Mel-28, WM983B, 1205Lu, and WM9. These cell lines were purchased from ATCC, Coriell, or were a generous gift from Dr. J. Alan Diehl or Dr. Alain Mauviel. All melanoma cell lines were cultured at 37 °C, 5% CO₂ in RPMI 1640 medium (HyClone, Logan, UT) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA), antibiotic–antimycotic (×100; ThermoFisher, Waltham, MA), and prophylactic plasmocin (InvivoGen, San Diego, CA) at 37 °C and in 5% CO₂. The HMLE human mammary cell line was obtained from Dr. Sendurai Mani and cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 5% calf serum, 0.5 μg/ml of hydrocortisone, 10 μg/ml of insulin, 20 ng/ml of EGF, and 1% antibiotic–antimycotic.
Stable cell lines were generated by lentiviral transduction with Polybrene (8 μg/ml; Sigma). 24 h post-transduction the medium was changed, and 48 h post-transduction the cells were selected and cultured with 0.125–0.5 μg/ml of puromycin (InvivoGen). Pools of stably transduced cells were analyzed. shRNA sequences are listed in Table 2. UV treatments were conducted using Fisher Scientific UV Cross-linker FB-UVXL-1000 at 0, 5, or 50 J/m² in room air, and then incubating in normal cell culture conditions for 24 h.

ImKCs (SCC119) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ImKCs were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) containing 1% antibiotic-antimycotic (100×) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific, Inc.) in an incubator at 37 °C under 5% CO₂ conditions.

The human high-grade chondrosarcoma cell line OUMS-27 [21 (link)], characterized by short tandem repeat analysis (access code; CVCL_3090), was obtained from Okayama University.
Cells were grown in supplemented Dulbecco’s modified Eagle medium with 10% (v/v) heat inactivated fetal bovine serum and 1% antibiotic-antimycotic (100×, Thermo Fisher Scientific, Waltham, MA, USA) under an atmosphere of 95% air and 5% CO₂ at 37 °C. Cells were confirmed to be free of mycoplasma infection using an e-Myco Mycoplasma PCR Detection Kit (iNtRON Biotechnology, Gyeonggi-do, Korea).