Propidium iodide

For the flow cytometric analysis sorting, skin cells were sorting according to Propidium Iodide (Biosharp, China). The PI-negative cells were immunolabeled with IL-20RB Polyclonal antibody (Proteintech, USA), then immunolabeled with Goat Anti Rabbit IgG(H&L)-Alexa Fluor 488 and PE anti-human CD140b (PDGFRB) Antibody (Biolegend, USA). IL20RB-positive and PDGFRB-positive cells were collected on Beckman MoFloXDP for RT-qPCR analysis of gene expression levels, the result was analyzed by FlowJo.
For flow cytometry, live DCs were immunolabeled with APC-CY7-livedead (Thermo Fisher Scientific, Massachusetts, USA), Brilliant Violet 605™ anti-mouse CD11C (BioLegend, CA), PE anti-mouse CD80 (Thermo Fisher Scientific), FITC anti-mouse CD86 (Thermo Fisher Scientific) at 4°C for 30 min. All cells were detected on Beckman Cytoflex LX and the result was analyzed by FlowJo.

The 6-well plates were divided into three groups, and 5.0 × 10⁵ cells (MDA-MB-231 and MCF-7) were added to each well. After adherence, added 2 mL of Syringin with the concentration of 0, 160, 320 µg/mL was added for 48 h, respectively. Then, the cells were fixed with 4% polyoxymethylene for 15 min. After washing with PBS 3 times, 1.0 mL Hoechst 33342 (Beyotime, 8 µg/mL) to each well for 15 min at 37 °C. Then, added 1.0 mL propidium iodide (PI) (Biosharp, 20 µg/mL) to each well at 4 °C for 10 min, and observed the cells with a fluorescence microscope.

LX-2 cells were preliminary serum-starved for 24 h in DMEM before Sjp40 treatment and synchronized in the G1 phase. And then the medium was replaced with DMEM containing 10% FCS with or without Sjp40 (20 μg/ml). Following stimulation cells were trypsinized and resuspended in 0.3 ml PBS, fixed with 0.7 ml anhydrous ethanol at 4 °C overnight. Fixed cells were resuspended in 0.9 ml PBS and incubated with Rnase A (50 μg/ml; TAKARA, Dalian, China) for 20 min at 37 °C. Cellular DNA was labeled with propidium iodide (50 μg/ml; Biosharp, Hefei, Anhui, China) for 30 min at 4 °C. Samples were filtered to remove cell clumps and analyzed by flow cytometry using a FACSCalibur instrument (BD Biosciences, San Jose, CA, USA).

For cell cycle analysis, cells were serum-starved for 72 h in DMEM, and then the medium was replaced with DMEM containing 10% FBS. After cultured for indicated time, the cells were harvested and fixed with ethanol at 4°C overnight. Subsequently, the cells were incubated successively with RNase A (Sangon, China) at 37°C for 30 min and propidium iodide (PI, Biosharp, China) on ice for 30 min. Then cell cycle analysis was performed using a flow cytometer from BD Biosciences (USA).

SL4 [(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-trifluoromethylphenyl)-propenone], of purity greater than 98%, was synthesized in the Medicine Chemistry Laboratory at Shenyang Pharmaceutical University (see Fig. 1A). The agent was dissolved in DMSO to 100 mM and stored at −20 °C. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was purchased from Sigma, U.S.A. and was dissolved in PBS. Propidium iodide (PI) was purchased from Biosharp and was dissolved in distilled water. SB203580 (a specific inhibitor of MAPK/p38), SP600125 (a specific inhibitor of MAPK/JNK) and PD98059 (a specific inhibitor of MAPK/ERK) were obtained from Sigma Aldrich (St Louis, MO). A83-01, a TGF-β inhibitor, was purchased from Tocris Bioscience (Minneapolis, MN). The primary antibodies against CyclinA2, CyclinB1, phospho-CyclinB1, cdc2, phospho-cdc2(Tyr15), p21, p53, Wee1, cdc25C, BRCA1, PCNA, Smad3, phospho-Smad3, Smad2, ERK1/2, phospho-ERK1/2, p38, phospho-p38, JNK, phospho-JNK, PARP, clv-PARP, and HIF-1α were purchased from Cell Signaling Technology (Danvers, MA); antibodies to β-actin, Smad4, Smad6, Smad7, and specific siRNA for HIF-1α were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).