Step one plus real time pcr assay

Cells were treated with 0.1% dimethyl sulfoxide (DMSO), 5 µg/ml lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, USA), or 5 µg/ml LPS+50 µg/ml BG for 6 hours and 24 hours. The dose of BG was determined by MTT assay. Total RNA was gained with an RNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesised from 3 µg of total RNA with a cDNA synthesis kit containing ImProm-II™ reverse transcriptase and oligo-dT primers based on protocol suggested by manufacturer (Promega, Madison, WI, USA). One microliter of cDNA was amplified using forward and reverse primers. The primers used in the study are shown in Supplementary Table 1. Electrophoresis with PCR products were done on a 1% agarose gel and visualized with ultraviolet light.
The real-time PCR was conducted with Step one Plus real-time PCR Assay (Applied Biosystems, Waltham, MA, USA). All reactions were conducted with Power SYBR Green premix (Applied Biosystems) using 50 ng cDNA and 10 pM of specific oligonucleotide primers. The primers used in the study are shown in Supplementary Table 1. Cycling conditions for amplification were as follows: 95℃ for 10 minutes and 40 cycles at 95℃ for 15 seconds and 60℃ for 60 seconds. The products of PCR were evaluated with the Step one Plus real-time PCR analysis software (Applied Biosystems).

Cells were treated with a variety of concentrations of PM10 (10 µg/ml, 25 µg/ml, 50 µg/ml, and 100 µg/ml) for 24 h. PM10 (PM10-like, European reference material ERM-CZ120) was obtained from Sigma-Aldrich. PM10 (100 µg/ml)-treated cells were also treated with 10 mg/ml of SHE for 24 h. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized from 3 µg of total RNA with a cDNA synthesis kit containing ImProm-II™ reverse transcriptase and oligo (dT) primers based on the protocol suggested by the manufacturer (Promega, Madison, WI, USA).
Real-time polymerase chain reaction (PCR) was conducted with Step One Plus Real-Time PCR Assay (Applied Biosystems, Foster City, CA, USA). All reactions were conducted with Power SYBR Green premix (Applied Biosystems) using 50 ng cDNA and 10 pM primers. The cycling conditions for amplification were as follows: 95℃ for 10 min, 40 cycles at 95℃ for 15 s, and 60℃ for 60 s. The products of the PCR were evaluated with the StepOnePlus Real-time PCR analysis software (Applied Biosystems). The primers used in the study are shown in Supplementary Table 1.

We treated the cells with 5 µM of dieckol or 100 µg/ml of PM10 for 24 h. We also treated the cells with 5 µM of dieckol and 100 µg/ml of PM10 simultaneously for 24 h. Subsequently, we obtained the total RNA using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and synthesized the cDNA from 3 µg of the total RNA with a cDNA synthesis kit containing ImProm-II™ reverse transcriptase and oligo-dT primers based on the manufacturer’s suggested protocol (Promega, Madison, WI, USA).
We conducted the real-time PCR with the StepOnePlus™ Real-Time PCR Assay (Applied Biosystems, Foster City, CA, USA) and all reactions with the Power SYBR Green premix (Applied Biosystems) using 50 ng of the cDNA and 10 pM of the primers (The PCR primer sequences are summarized in Supplementary Table 1).
The cycling conditions for amplification were as follows: 95℃ for 10 min and 40 cycles at 95℃ for 15 s and at 60℃ for 60 s. We evaluated the PCR products using the StepOnePlus (Applied Biosystems).