Penicillin and streptomycin solution

Que (purified ≥ 98%), kaempferol, myricetin, catechin, and GA were purchased from Sigma (St. Louis, MO, USA). Water used in this study was purified using the Milli-Q water purification system (WATER PRO^® PS, Missouri, USA). All other chemicals were analytical grade and were purchased from local chemical suppliers. HPLC-grade solvents obtained from Fisher Scientific (Seoul, Korea) were used. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco^® (Invitrogen, USA). HBSS trypsin–EDTA solution, and penicillin and streptomycin solution were purchased from GibcoTM (Life Technologies, Grand Island, NY, USA). Sodium pyruvate solution was purchased from Calbiochem^® (Merck, Germany). Fetal bovine serum (FBS) was purchased from Hyclone^® (GE Healthcare Life Sciences, Utah, USA).
Trizol reagent (Invitrogen, Camarillo, CA, USA) and the ReverseAid First-Strand cDNA Synthesis Kit (BioRad, Hercules, CA, USA) were used for RNA extraction and cDNA synthesis. Forward and reverse primers for the CYP450 genes were synthesized for mRNA expression level determination (Pacific Science Company, Bangkok, Thailand). Maxima SYBR Green qPCR master mix (Westburg Life Sciences, The Netherlands) was used for real-time PCR (RT-PCR) reactions.

RA (purified ≥ 98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Water used in this study was purified using the Milli-Q water purification system (WATER PRO^® PS, MO, USA). All other chemicals were of analytical grade and purchased from local chemical suppliers. HPLC-grade solvents obtained from Fisher Scientific (Seoul, Korea) were used for drug analysis. Dulbecco’s modified eagle’s medium (DMEM), HBSS Trypsin-EDTA solution, and Penicillin and Streptomycin solution were purchased from Gibco^TM (Life Technologies, Grand Island, NY, USA). Sodium pyruvate solution was purchased from Calbiochem^® (Merck, Germany). Fetal bovine serum (FBS) was purchased from Hyclone^® (GE Healthcare Life Sciences, Salt Lake, UT, USA). Twenty-four well cell culture plates were purchased from Costar (Corning Incorporated, Corning, NY, USA). Millicell^® Cell Culture inserts and Millipore Express^® PES Membrane (0.22 μm) were purchased from MILLIPORE (USA).

Mouse HPs were generated as previously described [31 (link)]. Briefly, primary adult mice hepatocytes were isolated from 8 to 12 weeks old female BALB/c mice by two-step perfusion method. After perfusion with Ca⁺² free Hank’s/EGTA solution and digestion with collagenase solution (Millipore Sigma), the digested livers were filtered and the suspension was collected via centrifugation at 50 g at 4 °C. Dead cells were removed, the remaining cells were washed twice and cultured with small hepatocytes basal medium (SHM) [DMEM/F12 (high glucose, Hyclone) supplemented with 5 mM HEPES (Sigma), 10 ng epidermal growth factor (PeproTech), 1% ITS (Gibco), 30 mg/L L-proline (Sigma), 0.05% BSA (Gibco), 10–7 M dexamethasone (Dex) (Selleck), 1 mM ascorbic acid, 10 mM nicotinamide (Stem Cell), and 1% penicillin and streptomycin solution (Life Technologies)] supplemented with 10% FBS (Gibco). Purified mouse hepatocytes were then seeded on collagen-coated plates in SHM with or without the combination of the small molecule inhibitors, Y-27632 (Selleck), 0.5 mM A-83–01 (Selleck) and CHIR99021 (Selleck). One day after seeding, the medium was changed and every other day thereafter. We confirmed all procedures for hepatic and biliary functions described for mouse hepatic progenitors as mentioned in [31 (link)].

RPMI 1640, penicillin and streptomycin solution, and fetal bovine serum (FBS) were purchased from Life Technologies Inc. TAK-242, an inhibitor of TLR4 (TLR4i), was obtained from Calbiochem (San Diego, CA, USA), respectively. Imatinib mesylate, a selective tyrosine kinase inhibitor, was obtained from Sigma. Anti-Bcl-2 antibodies (554160) were obtained from BD Biosciences. Anti-TLR4 (sc-10741), anti-ERK1/2 (sc-154), anti-β-actin (sc-47778), anti-pro-caspase 9 (sc-17784), anti-pro-caspase 3 (sc-7272), anti-phospho-AKT (sc-109903), anti-AKT (sc-8312), anti-PDGFRα (sc-398206), anti-S100A8 (sc-20174), anti-S100A9 (sc-20173), and anti-Ki-67 (sc-23900) antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz). Z-DEVD-fmk (60332), anti-phospho-STAT3 (9131), anti-STAT3 (9139), anti-cleaved caspase 9 (9501), anti-cleaved caspase 3 (9664), anti-AIF (4642), anti-Bax (2772), anti-Mcl-1 (4572), anti-phospho-ERK1/2 (9101), anti-phospho-p38 MAPK (9211), anti-p38 MAPK (9212), anti-phospho-JNK (9251), anti-JNK (9252), anti-rabbit IgG-HRP (7074), and anti-mouse IgG-HRP (7076) antibodies were procured from Cell Signaling Technology. Anti-phospho-Bad (9296) and anti-Bad (9292) antibodies were purchased from New England Biolabs. Anti-phospho-PDGFRα antibodies (ab5460) for immunohistochemistry were obtained from Abcam.

DMEM/F12, RPMI 1640, penicillin and streptomycin solution, trypsin/EDTA and TRIzol reagent were obtained from Life Technologies Inc. (Gaithersburg, MD, USA). Fetal bovine serum (FBS) was acquired from RMBIO (Missoula, MT, USA). Dermatophagoides pteronissinus (DP) extract was purchased from Cosmo Bio (Tokyo, Japan). TLR4 inhibitor (TLR4i), CLI-095, was purchased from Invivogen (San Diego, CA, USA). Inhibitors for PI3K (LY294002), AKT (AKTi), ERK (PD98059), p38 MAPK (SB202190), JNK (SP600125) and NF-κB (BAY-11-7085) were obtained from Calbiochem (San Diego, CA, USA). Antibodies against-phospho-AKT, AKT, ERK2, TLR4, and BCL2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-p38 MAPK, anti-phospho-ERK1/2, anti-phospho-JNK, anti-p38 MAPK, anti-JNK, anti-cleaved caspase 9, anti-cleaved caspase 3, BAX, and MCL-1 were procured from Cell Signaling Technology (Beverly, MA, USA).