Psoralen

RPMI-1640 medium (11 mM glucose and L-glutamine) and fetal bovine serum were purchased from Gibco (Paisley, UK). Penicillin/streptomycin antibiotic mixture and Dulbecco's phosphate-buffered saline (DPBS) were purchased from WELGENE (Daegu, Korea). 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Duchefa (Haarlem, Netherlands). Primary antibodies against poly(ADP-ribose) polymerase (PARP) (9542), caspase-3 (9662), bcl-2 (2876), bax (2772), p62 (5224S), and beclin-1 (3495) were purchased from Cell Signaling Technology (Beverly, MA, USA), and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit, sc-2004; anti-mouse, sc-2005) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Psoralen, isoPsoralen, palmitate, rapamycin, and bovine serum albumin were obtained from Sigma-Aldrich (St Louis, MO). Bakuchiol was purchased from Enzo Life Sciences Inc. (Farmingdale, NY).

Virulent VACV strain WR and the correspondent VACV mutant lacking B18R expression (VACVΔB18, [14 (link)]) were grown in BSC-1 cells and stocks of semipurified virus were prepared by sedimentation through a 36% sucrose cushion. L929 cells were infected with VACV or VACVΔB18 with a multiplicity of infection of 5 plaque forming units (pfu)/cell in order to ensure the infection of all cells to obtain a representative RNA-seq profile of each condition. After adsorption of virus for 1 h at 37°C, the virus-containing medium was removed, and cells were washed twice with phosphate-buffered saline and replaced with fresh culture medium supplemented with 2% fetal bovine serum. Infected cells were then incubated at 37°C and harvested at 4 or 8 h postinfection (hpi) by scrapping. Where indicated, IFN (50 units/ml) was added to the infected cultures at 4 hpi and the incubation extended at 37°C to 9 hpi. Inactivation of viruses was performed as previously described [18 (link)], by incubation with 2 μg/ml psoralen (4-9-aminomethyl-trioxsalen; Sigma) for 10 min and then UV-irradiated for 10 min with 2.25 J/cm² in a Stratalinker 1800. Complete inactivation (>10⁸-fold reduction in pfu) was confirmed by plaque assay in BSC-1 cells.

The MTT assay was conducted to evaluate whether psoralen (Aladdin Chemical Company, Shanghai, China) was toxic on hPDLCs. psoralen was dissolved in dimethyl sulfoxide (DMSO) (Sigma, USA). hPDLCs were collected by trypsinization and seeded into a 96-well microplate at a density of 10⁴ per well. After 24 h incubation, the cells were treated for another 24 h with psoralen at different concentrations of 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL and 25 μg/mL. Then 200 µl MTT (0.5 mg/mL) dilution was added to each well and the microplate was put in the incubator in the dark for 4 h. An equal volume of DMSO was added after extracting the MTT liquid, then the microplate was shaken for 10 min to dissolve the crystals. The absorbance at 490 nm was observed with microplate reader (Bio-Tek, USA).

Ultra-pure water (18 mΩ) was obtained from a Milli-Q water purification system (Millipore Co., Ltd., Milford, MA, USA). High-performance liquid chromatography (HPLC)-grade methanol, acetic acid, and 56 analytical standards, including catechinic, scopolin, chlorogenic acid, epicatechinic, vanillic acid, caffeic acid, purerarin, syringic acid, daidzin, glycitin, scopoletin, eriocitrin, umbelliferone, p-coumaric acid, dihydroquercetin, sinapic acid, genistin, liquiritin, ferulic acid, salicylic acid, rutin, isoferulic acid, m-coumaric acid, naringin, hesperidin, resveratrol, xanthotoxol, silydianin, sinapyl alcohol, o-coumaric acid, liquiritigenin, kaempferol, 2’-hydroxygenistein, eriodictyol, daidzein, psoralen, glycitein, quercetin, didymin, bergaptol, naringenin, luteolin, cinnamic_acid, hesperetin, genistein, bergapten, diosmetin, isoliquiritigenin, coumestrol, sinensetin, formononetin, medicarpin, imperatorin, biochanin A, tangeretin, and rotenone (displayed in Table S2), were purchased from Sigma-Aldrich Co., Ltd. The stock solutions of these authentic standards were 10.0 mg of each standard dissolved in 10 mL methanol. Then, the stock solutions were diluted to various concentrations before analysis. All stock solutions were sealed with Parafilm^® and stored in a −20 °C freezer.

Testosterone propionate (TP) was purchased from Wako Pure Chemicals (Tokyo, Japan). Finasteride was supplied from Merck & Co., Inc. (Kenilworth, NJ, USA). Antibodies against androgen receptor (AR, sc-816), proliferating cell nuclear antigen (PCNA, sc-56), E2F1 (sc-193), Rb (sc-377528), cyclin D1 (sc-753), and β-actin (sc-81178) were obtained from Santa Cruz Biotechnology, Inc. (TX, USA). Antibody against prostate-specific antigen (PSA, Catalog number, PB9259) was purchased from Boster Biological Technology (CA, USA). Loganin (≥97.0%), psoralen (≥99.0%), and isopsoralen (≥95.0%) were procured from Sigma-Aldrich (Seoul, Korea). Morroniside (≥97.0%) was purchased from ChemFaces (Wuhan, Hubei, China). HPLC-grade acetonitrile and water were procured from JT&Baker (Seoul, Korea).