Reagent

WST-1 assay kit was purchased from Daeillab (Daeillab, Korea). and dihydrotestosterone (DHT) were purchased from Sigma Aldrich (Sigma Aldrich, USA). Specific antibodies such as Bcl-2, Bax, APAF-1 was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). Specific antibodies such as t-mTOR, p-mTOR, LC3, p62 Cyclin E1, p-cdk2, t-p70 S6K, p-p70 S6K1, t-4E-BP1, p-4E-BP1 were obtained from Cell Signaling Technology (Beverly, USA). and Specific antibodies such as β-actin and Active-caspase-3 antibodies were purchased from Abcam (Cambridge, USA). Muse Cell Cycle Kit (MCH100106) and Muse Cell Analyzer (PB4455ENEU) were purchased from Millipore (EMD Millipore Corporation, Germany).

Extraction and fermentation of trapa japonica fruit

Trapa japonica fruit (Taxa and representative voucher specimen number: KP255650) was grown in China and purchased from BS corporation (BS corporation, KOREA). The hot water extract of Trapa japonica fruit was heated at 37 °C with distilled water, followed by concentration and vacuum filtration in the water bath at 37 °C. The hot water extract of Trapa japonica fruit (10,000 ppm) was fermented using Bacillus methylotrophicus from MRS agar medium and Bacillus subtilis from MRS agar medium (respectively 1.0 × 106 cfu/ml). Trapa japonica fruit and medium were mixed with glucose, yeast extract and soytone. The final fermentation of extract was performed by mixing microorganisms (Bacillus methylotrophicus and Bacillus subtilis). It was fermented in a 37 °C fermenter for 72 h, followed by filtration with a 0.2 μm filter and centrifugation to remove the microorganisms completely. The fermented Trapa japonica fruit extract was sequentially separated into hexane, CH2Cl2, EtOAc, N-BuOH fractions and water layers. Subsequently, the water layer was separated using the silica gel column (Waters, USA) to obtain the six fractions. The six fractions were resolved using a Sephadex-LH20 column (Waters, USA). Fractions 1–6 were obtained via HPLC prep HPLC Xterra C18 ODS column (Waters, USA). The fraction was refined using HPLC (Waters, USA). The mobile phase consisted of water:ACN:MeOH solution (water:MeOH = 90:10, 80:20, 70:30, and 0:100) pumped at a flow rate of 1 mL/min. The fraction 2 was chromatographed over a RP silica gel column (Waters, USA), and eluted using the water-MeOH gradient and monitored via TLC (thin layer chromatography) to separate the five fractions. Later, single compounds 1 to 6 were isolated from the fraction 2–2 using preparative HPLC. The compound 1 was concentrated under vacuum, and freeze-dried completely. The compound 1 powder was designated as ‘AC2 peptide’ and dissolved in distilled water.

Fractionation and isolation of AC2 peptide from Bacillus/Trapa japonica fruit and NMR profiling

Ferment Trapa japonica fruit was further fractionated with n-hexane, CH 2 Cl 2 , EtOAC, n-BuOH and then with water. Five fractions were obtained, respectively. After confirming the protein content, the effective Water fraction was concentrated under reduced pressure using a rotary vacuum concentrator (Dlab, RE100-pro). And then subjected to column chromatography on columns of silica gel (70~230 mesh, Merck art 4 * 20 cm) using a Water–MeOH gradient (Water: MeOH = 90:10, 80:20, 70:30, 0:100) and Sehpadex LH20 column chromatography (MeOH) to afford five fractions (Fr. 1~5). Fraction 2 was chromatographed over a RP silica gel column, eluting with a Water-MeOH gradient and monitored by thin layer chromatography (TLC Merck Art.) to separate five fractions (Fr. 2–1~2–5). After that, single compounds 1 to 6 were isolated from fraction 2-2 by high-performance liquid chromatography (HPLC) preparation. HPLC analysis was performed on a Waters 2690 Separation module and a Waters 2487 Dual Absorbance Detector. The analytical column (Xterra C18 ODS, 0.5 × 250 nm) was packed with Lichrospher 100RP-18 (15μm, Merck Co,) Compound 1 White amorphous powder; EI-MS m/z 303.13 [M] + 1H-NMR (D2O, 600 MHz) ▯1.859 (4 H, m, 13, 6-CH2), 1.941 (3 H, s, 15-CH3), 2.025 (1 H, m, 7-CH2), 2.138 (1 H, m, 7-CH2), 2.439 (2 H, m, 14 S-CH2), 3.818 (2 H, s, 2-CH2) 4.321,4.394 (1 H, m, 8-CH), 13C-NMR (D2O, 150 MHz) using Bruker Avance II+-500 FT-NMR Spectrometer (Bruker, USA).

Cell culture

Human dermal papilla cells were obtained from CEFO (CEFO, KOREA). Human dermal papilla cells were grown in DMEM medium (Hyclone, USA) containing 1% antibiotics (100 U/ml penicillin and 100 mg/streptomycin) and 10% Fetal bovine serum (Hyclone, USA) and at 37 °C in a 5% CO 2 atmosphere. Cells were suspended by Trypsin-EDTA (Hyclone, USA) every 2 days.

WST-1 assay

Cells were seeded at 3.8 × 105 cells/ml in a 12-well plate for 24 hours and were incubated with AC2 peptide (0.1–10 mg/ml) and DHT (dihydrotestosterone, 1 mg/ml) for various hours. Following incubation with the AC 2 peptide and DHT, the cells were incubated with a 100 μl/ml Wst-1 solution (Daeillab, Korea) for 60 min. Then, the optical densities of the solutions were quantified at a 450 nm wavelength by using a FLUOstar Omega (BMG labtech, Germany).

Determination of cell cycle

Cells were seeded at 9.5 × 105 cells/ml in a 6-well plate. After 24 hours incubation, cells were treated with AC2 peptide (0.1–10 mg/ml) and DHT (dihydrotestosterone, 1 mg/ml) for 24 hours. Following incubation, the cells were resuspended with PBS. And slowly add 200 μL of pre-cold 70% ethanol. After incubate for 24 hours at −20 °C, the fixed cells were mixed with 200 μL of premixed reagent including the nuclear DNA intercalating stain PI (propidium iodide) and RNAse and incubated for 30 minutes at room temperature in the dark. Then, the stained cells were analyzed in Muse Cell Analyzer (Merck Millipore Co.). the stained cells at different stages of the cell cycle, based on differential DNA content.

Western blotting

Cells were seeded at 1 × 106 cells/ml in a 6-well plate. After incubation, cells were treated with AC2 peptide (0.1–10 mg/ml) and DHT (dihydrotestosterone, 1 mg/ml) for 24 hours. After a 24 hours, cells were rinsed with pre-cold PBS, scraped with a RIPA buffer (Sigma Aldrich, USA) with Halt Protease and Phosphatase inhibitor cocktail (ThermoFisher, USA) and sonicated using Ultra sonicator (KP Tech., Korea) subjected to the western blot analysis. Protein quantification was performed using a Bradford assay and 50 μg of protein were loaded per lane. Primary antibodies (t-mTOR (1: 2,000; cat. No. #2972), p-mTOR (1: 2,000; cat. No. #2971), Raptor (1: 2,000; cat. No. 24C12), t-4E-BP1 (1: 2,000; cat. No. #9452), p-4E-BP1 (1: 2,000; cat. No. 236B4), t-p70-S6K (1: 2,000; cat. No. #9202), p-p70-S6K (1: 2,000; cat. No. #9234 T), Bcl-2 (1: 2,000; cat. no. #4223), Bax (1: 2,000; cat. no. #2772), APAF (1: 2,000; cat. No. #G1310), caspase-3 (1: 1,000; cat. no. ab4051), p62 (1: 2,000; cat. No. #88588), caspase (1: 2,000; cat. No. #G1310), Beclin-1 (1: 2,000; cat. no. #3738), LC-3 (1: 1,000; cat. #3868), Cyclin E1 (1: 2,000; cat No. 20808), p-Cdk2 (1: 2,000; cat ab76146) and β-actin (1: 2,000; cat. no. 3700)) reacted overnight at 4 °C and secondary antibodies (Anti-mouse IgG, HRP-linked Antibody #7076 and Anti-Rabbit IgG, HRP-linked Antibody #7074) reacted for 120 min at 4 °C. Western blotting was repeated at least times for each experiment.

Immunoprecipitation

Cells were seeded at 1 × 106 cells/ml in a 6-well plate. After a 24 hours incubation, cells were treated with AC2 peptide (0.1–10 mg/ml) and DHT (dihydrotestosterone, 1 mg/ml) for 24 hours. After a 24 hours, cells were rinsed twice with ice cold PBS, scraped with a RIPA buffer (Sigma Aldrich, USA) with Halt Protease and Phosphatase inhibitor cocktail (ThermoFisher, USA). Cells were centrifuged at 14,000 x g for 5 minutes to obtain cell lysates. Cell lysates were incubated with anti-mTOR antibody (Cell signaling, USA) and reacted overnight at 4 °C. Then, pretein A/G PLUS-agarose (Santa Cruz Biotechnology, USA) was added to the cell lysate and reacted overnight at 4 °C. The next day, the beads were washed with ice cold PBS three times and heated with SDS sample buffer for 5 minutes at 100 °C. The samples were subjected to the western blot analysis. Western blotting was repeated at least times for each experiment.

Organotypic 3D cell culture model

The orgranotypic 3D culture (OTC) is followed Hiro Nakagawa, MD, PhD protocol36. the methods are followed.

Matrix (consist of Fetal bovine serum (Hyclone, USA) and DMEM medium (Hyclone, USA), type I collagen) is prepared along with Human Dermal papilla cells on overnight. Human Dermal papilla cells were seeded on 4 days, grown in a EP2 medium (consist of DMEM, F12, Progesterone, L-Glutamine, Hydrocortisone, Newborn Calf Serum etc.), and then exposed for 5 days to reduced volume of medium. Then, change the medium to EP3 medium (EP2 medium without Progesterone) for 5 days. Incubated for 10 days with AC2 peptide (0.1–10 mg/ml) and DHT (dihydrotestosterone, 1 mg/ml) at 37 °C in a 5% CO 2 atmosphere.

Immunohistochemistry (IHC)

The organotypic 3D cell culture models were fixed in 10% Neutral buffered formalin for overnight and incubated with PBS at 4 °C for 2 days. Then, the models were sectioned and embedded in paraffin. The embedded sections were fixed in cold-acetone for 10 min. the sections were treated with 3% H 2 O 2 and blocked with 10% normal goat serum (Invitrogen, USA). Then, the sections were incubated with a Ki-67 primary antibody (1:400; cat No. #9027) overnight at 4 °C. On the second day, the sections were incubated with an Alexa Flour 488-conjugated secondary antibody (1:1000; cat No. #4412) and mounted using Antifade mounting medium with DAPI (VECTASHIELD, UK) for 2 hours. the sections were observed using a confocal microscope (Olympus, Japan).

AC2 peptide synthetic mechanisms

AC2 synthesized three amino acids (Fmoc Serine, Glycine and Proline) through the following process. First, three amino acids were inflated and cleaned using 2-Chlorotrityl chloride resin and methylene chloride (MC). Then, after reacting with DMF (Dimethylamide) and DIPEA (Dimethylamine) in amino acids, the primary amino acid was attached by reacting with a De-Blocking solution. next, dissolving with HOBt containing DMF and mixing with HBTU added DIPEA to attach another amino acid. The synthesized resin was concentrated with TFA (Tripluoroacetic acid) and MC. The synthesis process was repeated to react with TFA and distilled water in concentrated resins, distributed in ether, and dried. Dried powder was dissolved in distilled water to synthesize AC2 peptide with a purity of 95% or higher using prep LC.

Statistical analysis

All the experiments were repeated at least three times and analyzed using t-tests (SPSS 20.0, USA). p <0.05 was considered to indicate a statistically significant difference.

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