Staining star

The inoculum described above was centrifuged at 13,000 × g for 5 min and the pellet was used for genomic DNA extraction using PrepMan™ Ultra Sample Preparation Reagent (Thermo Scientific, Waltham, USA) and PCR inhibitor removed by OneStep™ PCR inhibitor Removal Kit (Zymo Research, Irvine, USA). Target genes and PCR primer sequences are listed in Supplementary Table S1. The PCR reaction was composed of extracted DNA, primer pairs for each target gene, PCR-grade water, and Takara Ex Taq version 2.0 (Takara, Kusatsu, Japan). The PCR cycle was as follows: initial denaturation at 95 °C for 10 min, then 30 cycles of 1) Denaturation at 95 °C for 15 s, 2) Annealing and extension at 60 °C for 30 s. PCR products were separated by electrophoresis on 0.8% agarose gel in TAE buffer (40 mM Tris-HCl, 40 mM acetate, 1.0 mM EDTA), stained with Staining STAR (DyneBio, Seongnam, Korea) and confirmed with Gel Doc^TM EZ Imager (Bio-Rad, Richmond, USA).

BMDCs (2 × 10⁶ cells/well) were treated for 24 h with 14BME20 or LPS. Total RNA from the BMDCs was extracted with RiboEX total RNA kit (GeneAll Biotechnology, Seoul, Korea) and reverse transcribed into cDNA using a RocketScript™ Reverse Transcriptase kit (Bioneer Corporation, Daejeon, Korea). After cDNA was synthesized, the cDNA was amplified by PCR using AccuPower® PCR PreMix (Bioneer Corporation). The sequences of the RT-PCR primers used in this study were as follows: murine TGF-β forward, 5′-TAT AGC AAC AAT TCC TGG CG-3′ and reverse, 5′-TCC TAA AGT CAA TGT ACA GC-3′; murine IL-10 forward, 5′-AGA AAT CAA GGA GCA TTT GA-3′ and reverse, 5′-CTG CAG GTG TTT TAG CTT TT-3′; murine IDO forward, 5′-TTA TGC AGA CTG TGT CCT GGC AAA CTG-3′ and reverse, 5′-TTT CCA GCC AGA CAG ATA TAT GCG GAG-3′; murine COX-2 forward, 5′-GTG GAA AAA CCT CGT CCA GA-3′ and reverse, 5′-TGA TGG TGG CTG TTT TGG TA-3′; murine IL-12p40 forward, 5′-TTA TGC AAA TTG TGA GCT TG-3′ and reverse, 5′-CCT TTG CAT TGG ACT TCG GTA G-3′; murine β-actin forward, 5′-CGC AGA GTC TCG CCA TTA TG-3′ and reverse, 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′. After cDNA amplification, the products were separated on 1.5% (w/v) agarose gels and stained with StainingSTAR (DyneBio, Gyeonggi-do, Korea). The relative expression of cytokines was analyzed by Image J software.