Skim milk

The liver tissue and HepG2 cells were lysed by RIPA lysis solution (Beyotime, China) to extract the protein. The protein concentration was detected by BCA reaction kit. After quantitative analysis, the total protein was denatured in this study. SDS-Page gel was used for electrophoresis, electrophoresis apparatus (Bio-RAD, USA) was adjusted to 120 V for electrophoresis, PVDF membrane (Millipore, USA) was used for membrane transfer, and skim milk (Sigma, USA) for blocking. Primary antibodies (Abcam, UK): Sirt1 (1:1000; ab189494), optic atrophy 1 (Opa1, 1:1000; ab157457), mitofusin 2 (Mfn2, 1:1000; ab124773), Drp1 (1:1000; ab184247), NRF1 (1:1000; ab34682), mitochondrial transcription factor A (TFAM, 1:1000; ab252432; Abcam; UK), Bcl-2 (1:2000; ab182858), cleaved-caspase 3 (1:500; ab2302), BCL2-associated X protein (Bax, 1:1000; ab32503), cleaved-caspase 9 (1:2000; ab32539) and GAPDH (1:2500; ab9485) were then added overnight to incubate. The next day, goat anti-rabbit antibody (1:2000; ab288151) was incubated for 1 h with slow shaking at 25 °C. The immunoreactive bands were visualized by intensive chemiluminescent reagent. The gray value was analyzed by ImageJ software.

For Western blot analysis proteins of the AH were extracted with sodium dodecylsulfate (SDS) lysis buffer (25 mm Tris–HCl, pH 6.8, 2.3% SDS, 10% glycerol, and 5%β‐mercaptoethanol). Protein extracts were separated on a 7–12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked in Tris-buffered saline with Tween 20 (20 mm Tris, pH 7.4, 150 mm NaCl, and 0.05% Tween 20) containing 5% skim milk (Sigma) for 1 h at room temperature and incubated overnight with anti-rabbit EZH2 (1:1000; Cell Signaling Technology), anti-rabbit β-actin (1:2000; Cell Signaling Technology), anti‐rabbit H3K27me2 (1:2000; Millipore), or anti‐rabbit H3K27me3 (1:2000; Millipore) antibodies at 4°C. The membranes were washed and then incubated with anti-rabbit IgG horseradish peroxidase-conjugated antibody (1:5000; GE Healthcare) at room temperature for 1 h. A chemiluminescent signal was detected using SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology) by the G:BOX chemi XRQ gel-imaging system (Syngene, Synoptics Ltd.), and densitometric analysis was performed using Quantity One 1-D analysis software (Bio-Rad).

The expression levels of TNF‐α, IL‐6 and TGF‐β were measured with ELISA using specific antibody (Santa Cruz Biotechnology, Dallas, TX, USA; Abcam, Cambridge, MA, USA). One hundred microliters of diluted antigen (rat serum, 2 μg·μL⁻¹ in phosphate‐buffered saline; PBS) was immobilized in a 96‐well microtiter plate (Nunc, Rockilde, Denmark) overnight at 4 °C. After incubation, the plates were washed three times in washing buffer (twice with PBS, once with Tris‐buffered saline and Polysorbate 20). One hundred microliters of blocking solution [5% skim milk (Sigma‐Aldrich) in PBS] was added to each well. Afterwards, 100 μL of primary antibody (1 : 1000; Santa Cruz Biotechnology, Abcam) in blocking solution was added, followed by incubation for 2 h at room temperature on a shaker. Plates were washed as before. One hundred microliters of secondary antibody‐tagged horseradish peroxidase (1 : 1000; Santa‐Cruz Biotechnology) in blocking solution was added to each well. The plates were incubated for 1 h at room temperature. Finally, the plates were washed four times and 50 μL of tetramethylbenzidine (Sigma‐Aldrich) was added. The color intensity of the solution was read in a microtiter reader at 650 nm.

Dimethyl sulfoxide (DMSO), α-MSH, NaOH, MTT, arbutin, synthetic melanin, Triton X-100, radioimmunoprecipitation assay (RIPA) buffer, skim milk, Tween 20, L-DOPA, and fucoxanthin analytical standard were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, trypsin-ethylenediaminetetraacetic acid, TRIzol solution, and bicinchoninic acid (BCA) protein assay kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against CREB, p-CREB, β-actin, and anti-rabbit horseradish peroxidase antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Primers against Tyr, Trp-1, Dct, Mitf, Mc1r, and GAPDH genes were synthesized by Bioneer (Daejeon, Korea). An enhanced chemiluminescence (ECL) kit and polyvinylidene fluoride (PVDF) membrane were obtained from Bio-Rad (Hercules, CA, USA). EtOH and EtOAc from DaeJung Chemicals and Metals Co., Ltd. (Siheung, Korea) were analytical grade.

For IHC staining of endothelial colonies on OP9 coculture plates, the cultures were fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature (RT). After washing, the samples were blocked with 2% skim milk (Sigma) in 0.1% Triton (Sigma) PBS solution (PBSMT solution) for 30 minutes at RT and then stained with 1:100 rat anti-mouse CD31 (BD Pharmingen, clone MEC 13.3) or rat anti-mouse Flk1 (BD Pharmingen, clone Avas 12α1) Ab in PBSMT at RT for 2 hours or at 4°C overnight. Next the plates were stained with 1:200 alkaline phosphatase-conjugated donkey anti-rat IgG secondary Ab (Jackson ImmunoResearch) in PBSMT at RT for 2 hours or at 4°C overnight. The colonies were visualized by VECTOR Blue Alkaline Phosphatase (Blue AP) Substrate Kit.
For culture immunofluorescence staining of murine EC culture, fixed cultures were blocked with 10% goat serum in 0.5% triton PBS solution (blocking solution) and sequentially stained with primary Ab (1:100 rat anti-mouse CD31, clone MEC 13.3) and secondary Ab (1:200 Alexa Fluor 488- or 647-conjugated goat anti-rat IgG; Cell Signaling Technology) in blocking buffer. All culture pictures were visualized using Leica DM IL microscope with a SPOT RT3 camera (Spot Imaging).

ELISA plates were coated with 400 ng/well purified TSG MAb in carbonate–bicarbonate buffer (pH 9.6) (Sigma, St. Louis, MO, USA, C3041) and held at 37 °C for 1 h. TSG was used to capture VLPs (or mutated VLPs) for antibody characterization because its binding epitope was excluded from the neutralizing sites. The plates were washed three times with PBST (PBS with 0.05% Tween-20, pH 7.3) followed by blocking with 0.5% skim milk (Sigma, 70166) in PBS for 30 min. After the removal of the blocking buffer, 1 µL of cell lysates containing VLPs (or mutated VLPs) was added at a dilution of 1:100 and incubated at 37 °C for 1 h. After washing three times, the conjugates (100 ng/well) were incubated for 1 h. After the final washing, freshly prepared TMB substrate (BD biosciences, Franklin Lakes, NJ, USA, 555214) was added and allowed to stand for 15 min. The reaction was stopped with 1 N HCl and measured at 450 nm with a Sunrise^TM Absorbance Reader (TECAN, Männedorf, Switzerland). Each test was performed in triplicate.