Amphotericin b

Human diploid fibroblasts (HDFs) were obtained from the ATCC (PCS-201-010, Manassas, VA, USA). Cells were cultured in the growth media composed of DMEM (Welgene, Korea) supplemented with 10% FBS (Welgene), Antibiotic-Antimycotic solution (100 units/ml penicillin, 100 ug/ml streptomycin and 250 ng/ml amphotericin B, Welgene) at 37 °C in 5% CO₂ incubator. We used senescent cells of which the population doubling time (DT) is more than 14 days and non-senescent cells of which DT is less than one days [25 (link)]. We used SIPS models of which non-senescent HDFs were treated with 20 μM etoposide for two days, or treated with 400 μM H₂O₂ for six days.

MC3T3-E1 is a pre-osteoblastic cell line established from newborn mouse calvarias. We purchased from the American Type Culture Collection (ATCC). Cells were cultured in α-minimum essential media (α-MEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS, WELGENE, Daegu, Korea), 100 units·mL⁻¹ penicillin G sodium, 10 μg·mL⁻¹ streptomycin, and 25 μg·mL⁻¹ amphotericin B (WELGENE, Daegu, Korea) at 37 °C in a humidified atmosphere with 5% CO₂.

RAW 264.7 cells (an immortalized murine macrophage cell line; ATCC, USA) were cultured in Dulbecco’s modified Eagle’s medium (WelGene Inc. Korea) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (WelGene Inc. Korea), 100 unit/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B (WelGene Inc. Korea), in 5% CO₂ at 37.5 °C. Cells were cultured in antigen free media in order to detect native protein expressional changes induced by 4HR.
About 70% confluent RAW 264.7 cells grown on Petri dish surfaces were treated with 10 µg/mL 4HR (safe single dose in dogs 100–300 mg/kg according to WHO food additives Series 35, 835) for 8, 16, or 24 hours; control cells were treated with 1 mL of normal saline. Cultured cells were harvested with protein lysis buffer (PRO-PREP^TM, iNtRON Biotechnology INC, Korea), and immediately preserved at −70 °C until required.

The human pancreatic cancer cell lines PANC-1 and Capan-1 were purchased from American Type Culture Collection (Manassas, VA, USA) and Korean Cell Line Bank (KCLB; Seoul, Korea), respectively. Human pancreatic stellate cells (PSCs) and a human monocytic cell line (THP-1) were obtained from ScienCell (HPaSteC; 3830, Carlsbad, CA, USA) and KCLB, respectively. Capan-1 and THP-1 cells were maintained in RPMI-1640 medium (Gibco, Grand Island, NY, USA), whereas PANC-1 and PSCs were maintained in high-glucose Dulbecco’s modified Eagle medium (HyClone, Logan, UT, USA). All media were supplemented with 100 μg/mL streptomycin, 100 units/mL penicillin, 250 ng/mL amphotericin B, and 10% fetal bovine serum (Welgene, Daegu, Korea). THP-1 cells were induced to differentiate into M2 macrophages (TAMs) using 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich, St. Louis, MO, USA) and 20 ng/mL of interleukin-4 (PeproTech, Cranbury, NJ, USA) as confirmed by CD-206 expression. Cell culture was maintained in a humidified atmosphere (5% CO₂/95% air) at 37 °C.

PC-3 and MDA-MB-231 cells were maintained in RPMI 1640 medium (Gibco-BRL, Paisley, UK) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL, Paisley, UK) and Antibiotic Antimycotic Solution (100 units/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B; Welgene, Daegu, South Korea) in humidified 5% CO₂/95% air at 37°C. MC3T3-E1, a mouse osteoblastic cell line, was maintained in growth medium (α-MEM; Gibco-BRL, Paisley, UK) supplemented with 10% FBS and Antibiotic Antimycotic Solution. Cells were cultured for 24 h at a density of 1 × 10⁵ cells/mL in 10 cm culture dishes or 24-well plates (Corning Inc., Corning, NY, USA). After reaching confluence, cells were cultured in differentiation medium supplemented with 50 mg/mL ascorbic acid and 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), and the medium was changed every 3 days. The 5-week-old male athymic nude mice (BL-6/Nu; Orient Bio Co. LTD, Seoul, South Korea) were housed under controlled conditions of light and fed ad libitum. All experimental procedures involving animals were performed in compliance with institutional and governmental requirements and approved by the Institutional Animal Care and Use Committee (CIACUC2017-A0002) of Chosun University, Gwangju, South Korea.

RAW 264.7 cells (an immortalized murine macrophage cell line; ATCC, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (WelGene, Inc. Korea) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (WelGene, Inc. Korea), 100 unit/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B (WelGene, Inc. Korea) in 5% CO₂ at 37.5 °C. Cells were not stimulated with bacterial antigen (LPS) to detect native protein expressions induced by the coffee extract.
RAW 264.7 cells were separately treated with different doses of dialyzed coffee extract (DCE) equivalent to 2.5, 5, or 10 cups of coffee (DCE-2.5, DCE-5, and DCE-10, respectively); control cells were treated with 1 mL of normal saline. Cells were incubated for 12 hours, harvested with protein lysis buffer (PRO-PREP^TM, iNtRON Biotechnology, INC, Korea), and immediately preserved at −70 °C in a deep freezer until required.

BEAS-2B cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Human bronchial epithelial (HBEL)/p53i cells were kindly provided by Dr. John Minna (University of Texas Southwestern Medical Center, Dallas, TX, USA)^{13 (link)}, and 1799, 1198, and 1170-I cells were kindly provided by Dr. Curtis C. Harris (National Cancer Institute, USA)^{14 (link)}. These cells were cultured in K-SFM (Thermo Fisher Scientific, Waltham, MA, USA) with 5 ng/mL recombinant epidermal growth factor (EGF) and 50 μg/mL bovine pituitary extracts. MLE-12 murine alveolar epithelial cells, THP-1 human monocytes/macrophages, and Wi38 human lung fibroblasts were purchased from ATCC. MLE-12 cells were cultured in HITES medium [DMEM-F12 media (Welgene, Inc., Gyeongssan-si, Republic of Korea) containing 1× insulin-transferrin-selenium solution (Thermo Fisher Scientific), 10 nM hydrocortisone, 10 nM β-estradiol, 10 mM HEPES, and 2 mM L-glutamine (Welgene)] with 2% fetal bovine serum (FBS) (Welgene) and 1× antibiotic-antimycotic solution (100 units/mL penicillin, 10 mg/mL streptomycin sulfate, and 25 μg/mL amphotericin B in 0.85% NaCl solution; Welgene). THP-1 and Wi38 cells were cultured in RPMI 1640 medium and DMEM, respectively, with 10% FBS and 1× antibiotic-antimycotic solution. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂.