MS medium (MSP09), 2,4 D (D001 and gelrite (G024) were purchased from Caisson Lab (USA). Tween 20 (P1379), gallic acid (G7384) kojik acid (K3125), α-MSH (M4135) and L- DOPA (D9628) were obtained from Sigma Aldrich (St Louis, MO, USA).
MTT (A2231) and DMSO (A3672) were purchased from Applichem (Darmstadt, Germany). DMEM (41966-029), IMDM (21980-032) and FBS (10270-106) obtained from Gibco Life Technologies (Carslbad, CA, USA).
RIPA buffer and RNA isolation kit and cDNA synthesis kit were purchased from respectively Thermo Fisher Scientific (Waltham, MA, USA), Invitrogen (Carlsbad, CA, USA) and Applied Biosystems (Forster City, CA, USA). BCA assay kit and Blocking kit (iBlot Transfer Stacks, nitrocellulose) and chromogenic detection kit (Western Breeze Chromogenic kit) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). ACTB (Sc-47778), TYR (Sc-20035), TRP1 (Sc-166857), TRP2 (74439) and MITF (Sc-56725) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Plant Cell Culture
Glycine max (soybean) seeds were surface-sterilized for 20 min in a 20% solution of commercial bleach containing 1-2 drop Tween 20, followed by 4 times rinse with sterile distilled water and incubated onto solid MS medium in the dark at 25 ºC for the germination. After 7 days, the hypocotyl explants were cut from germinated seedling and place onto solid MS medium supplemented with 1mg/L 2,4 D and 30 g/L sucrose. Friable calluses were used to establish cell suspension culture. Cell suspension culture was initiated by agitation 4 g of callus in 250 ml flasks containing 50 ml of liquid MS medium supplemented with 1 mg/L 2,4 D and 30 g/L sucrose at a shaking speed of 110 rpm. Cells were maintained by subculturing to fresh medium once every 14 days.
The cells were filtered and rinsed with distilled water. The fresh cells were lyophilizated. For the extraction 20 gram of the lyophilized cells were incubated with 200 ml 70% ethanol placed in the rotary shaker for 24 hours at room temperature. The extract were centrifuged at 10000 rpm for 10 minutes, and supernatant was filtered through PTFE syringe filter (0.22 µM). The fitrate was evaporated at 40 ºC under reduced pressure.
Determination of total phenolic content
The concentrations of phenolic compounds in the extract of soy cell culture were measured according to Folin Ciocalteu reagent method as decribed by Ainsworth and Gillespie . Briefly, 0,1 ml of soy cell culture extract (1 mg/ml) and 0,2 ml of Folin-Ciocalteu’s reagent mixed together. Then, 0,8 ml of 700 mM Na2CO3 was added and the reaction mixtures were incubated for 2 h at the room temperature in the darkness. The absorbance was measured at 765 nm with a spectrometer. A dose response linear regression was generated by using the gallic acid standart absorbance (100-500 µM) and the levels in the extract were expressed as gallic acid equivalents (µM of GAEs/g of extract).
Cell viability assay
B16-F10, mouse melanoma cells were cultured in DMEM supplemented with 10% (v/v) of FBS at 37 ºC in a humidified atmosphere containing 5% CO2. Cell viability was determinated using the MTT assay to the method described by Mosmann . B16F10 cells were plated into 24 well plate at a density of 0,7 x105 cells per well and incubated 24 hours. After the cells were treated with different concentrations of soy cell culture extract (SCE) for 48 h, then treated with MTT solutions (5 mg/ml) for 3 h after which the precipitated formazan was dissolved by DMSO. The optical density was measured at 570 nm with a microplate spectrophotometer.
Melanin contents assay
Cellular melanin content was determinated using a minor modification of a method described by Jin et al. . Briefly, B16F10 cells were seeded at a density 1x105 cells/well on the six well plate and, after 24 h of incubation the cells were treated with 100 nm α-MSH for 24 hours except control group. After treated with α-MSH, the medium was removed and the cells were treated with of SCE and kojic acid for 48 h. After incubation, pellets were dissolved in 1 N NaOH containing 10% DMSO for 1 hour at 80 ºC. The optical density of homojenizates were read at 475 nm using a microplate reader.
Cellular tyrosinase activity assay
The effect of SCE on cellular tyrosinase activity assay α-MSH induced tyrosinase activity in B16F10 cells were investigated following a slightly modified previous method . Briefly, B16F10 cells were seeded at a density 1x105 cells/ well on the six well plate and, after 24 h of incubation the cells were treated with 100 nm α-MSH for 24 hours except control group. After treated with α-MSH, the medium was removed and the cells were treated with of SCE and kojic acid for 48 h. After then the cells washed with PBS and lysed with including 1% Triton X-100 and 100 µM PMSF in PBS and centrifuged at 17500 g for 10 min at 4 ºC. Each 90 µl supernatant and 10 µl L-Dopa (2 mg/ml) was combined in the well of 96-well plate and incubated for 30 min at 37 ºC.
The oxidation of L-DOPA to dopachrome was measured at 475 nm with the absorbance reader.
Total RNA isolation and RT-qPCR analysis
B16F10 melanoma cells were cultured at a density of 2,5x105 in 25cm2 flasks and, after 24 h of incubation the cells were treated with 100 nm α-MSH for 24 hours except control group. After treated with α-MSH, the medium was removed and the cells were treated with of SCE for 48 h. Total RNA was isolated by using Pure RNA mini kit (Life Technologies) and total RNA was converted to cDNA using a cDNA kit according to manufacturer’s protocol (High Capacity cDNA Revers Transcription Kit, Applied Biosystems). 100 ng of cellular RNA was reverse-transcribed into cDNA. RT-qPCR analysis were performed with TaqMan probes (ACTB; Assay ID: 316568, TYR; Assay ID: 316568, TRP1; Assay ID: 316563, TYRP2; Assay ID: 317497 and MITF; Assay ID: 315218, RealTime Ready Custom Single Assays, Roche) using a RT-qPCR system (LightCycler 480, Roche). Data were analyzed using the 2[−△△ct] method  and presented as the means ±SD normalized to ACTB.
Western blot analysis
B16F10 cells were treated with SCE and kojic acid for 48 h after treatment with 100 nM α-MSH for 24 h. The proteins were isolated with RIPA buffer and amounts of total protein was determinated with BCA assay kit (Thermo Fisher Scientific Inc., USA). Then the proteins were separated by SDS page electrophoresis using %4-12 Bis-Tris gel. Following electrotransfer to nitrocellulose membranes with dry blotting (iBlot2 Dry Blotting System, Thermo Fisher Scientific) the membranes were blocked according to manufacturer’s protocol (Western Breeze Chromogenic kit, Thermo Scientific). Then the membranes incubated overnight with primary antibodies (ACTB; 1:1000, TYR ;1:50, TYRP1; 1:50, TYRP2; 1:100; MITF; 1:50) at 4 ºC and secondary antibody. The membrans were incubated with chromogenic substrate until the bands developed on the memrane.
Graphpad Prism version 8.3 was used for the statistical analysis of in vitro cytotoxicity, melanin content, cellular tyrosinase activity and RT-qPCR experiments. Data were expressed as the mean ± SD values of last three independent experiments. Statistical significance was analyzed using One-Way ANOVA and Tukey’s test and a P value <0,05 was considered to indicate statistical significance.