Arvo x3

For the evaluation of protein expression changes, 4.5 × 10⁵ 293FT cells were plated in 6-well plates. After 24 h, 0.72 μg of the let-7–overexpressing vectors were transfected using PEI (2.52 μl) and 200 μl of Opti-MEM. After 24 h incubation, CoCl₂ was added at a final concentration of 250 nM, and the plates were incubated for 24 h. We collected the proteins with RIPA buffer and performed a Western blot using the protocols described above. The experiment was performed for two times and the signal ratio compared with beta-actin was calculated. For sensor assay, 6 × 10⁴ 293FT cells were plated in 24-well plates. After 24 h, 210 ng of vectors expressing NC, WT LIN28A, or ZFm LIN28A; 60 ng of vectors expressing the let-7 sensor; and 60 ng of vectors expressing Renilla with SV40 promoter were transfected using 1.89 μl of PEI and 200 μl of Opti-MEM. After incubating for 24 h, CoCl₂ was added at a final concentration of 250 nM. After 24 h, 180 μl of firefly reagents were added, and the plates were incubated in the dark for 10 min. The luminescence was measured using ARVO X3 (PerkinElmer). We added the same amount of Renilla reagent again and after incubating the plates for 10 min in the dark and measured the luminescence using an ARVO X3 (PerkinElmer). The experiment was performed for three independent samples.

The melanin content of cultured cells was measured as described previously [14 (link)]. Briefly, melanoma B16 cells were seeded at a density of 2 × 10⁴ cells/well in 6-well culture plates and incubated at 37°C under 5% CO₂ atmosphere for 24 hours. The cells were then treated with or without IL-1β (2 μg lyophilized was diluted with 2 ml sterile distilled water and diluted with complete medium to the working concentration of 4 pg/μl, 200-01B-2, PEPROTECH, USA) for 48 hours, followed by washing twice with PBS and dissolving in 120 μl of 1 N NaOH (Solarbio, China) containing 10% DMSO (D12345, Gibco, USA) at 80°C for 2 hours. Afterwards, melanin content in homogenates was determined by measuring absorbance at 405 nm using a plate reader (ARVOTM X3; PerkinElmer, Waltham, MA, USA). The amount of cellular melanin was normalized to the number of cells in the samples.

Evaluation of intracellular calcium flux by PRRs ligands in TLR2-, TLR4-, NOD1-, and NOD2-knockdown PIE cells were performed according to the method of Murofushi et al. [36 (link)], with some modifications. Briefly, the knockdown cells (1 × 10⁵ cells/100 μL medium) were plated on 96-well Cell Culture Clear Bottom Black Plates (Corning Inc., New York, NY, USA) at 37 °C for 24 h. After the careful elimination of the medium by aspiration, 100 μL lording buffer of Calcium Kit-Fluo 4 (Dojindo Laboratories, Tokyo, Japan) was added and incubated at 37 °C for 1 h. The medium was then replaced by 100 μL of the recording medium. The dye-stained cells were washed with PBS and stimulated with 10 μL (1 mg/mL) of LTA, LPS, DAP, or MDP by the dropping system in a fluorescence spectrophotometer (ARVO^TM X3, PerkinElmer Inc., Billerica, MA, USA). The fluorescence intensity was recorded up to 140 s after stimulation.