Gotag qpcr master mix

Quantitative RT-PCR was performed to detect the levels of PP2Acα mRNA using GoTag qPCR Master mix (Promega, Madison, WI). Primer sequences are listed in following: PP2Acα, 5’-TCGTTGTGGTAACCAAGCTG-3’ (F), 5′-AACATGTGGCTCGCCTCTAC-3’ (R); GAPDH, 5’-GAAATCCCATCACCATCTTCCAGG-3’ (F), 5’-GAGCCCCAGCCTTCTCCATG-3’ (R).

Total RNA from roots and leaves was extracted using TRizol reagent (Sigma Aldrich, Belgium) according to the manufacturer’s instructions. RNA concentration was determined using a Quantus fluorometer (Promega, Netherlands). cDNA of each RNA template was synthesized using the iScript^TM kit (Bio-Rad, Belgium) and diluted five times with nuclease-free water. The quantitative reverse transcription PCR (RT-qPCR) assay was performed using a CFX96 Tough Real-time PCR Detection System (Bio-Rad, Belgium). Each PCR reaction contained 6.3 μL Gotagq^®PCR master mix (Promega, Netherlands), 2 μL cDNA, 0.6 μL each primer (5 μM), and 0.2 μL CXR dye (Promega, Netherlands), and 2.3 μL nuclease-free water. The thermal program was set up as follows: 95°C for 3 min; 39 cycles of 95°C for 10 s, and 60°C for 30 s, followed by a melting curve acquisition from 65 to 95°C with the rate of 0.5°C s^–1. The primers used for all target genes are shown in Supplementary Table 1. Elongation factor 1α (EF-1α) and β tubulin (β-TUB) primers were used as housekeeping genes. Gene expression analysis was done using qBase⁺ software (Biogazelle, Zwijnaarde, Belgium). Fold change was computed by diving the CNRQ values (calibrated normalized relative quantities) of the treated samples by values of the control samples. In each gene, four biological replicates and two technical replicates were done.

Chromatin immunoprecipitation (ChIP) was performed using SimpleChiP^® Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer’s protocol. In all, 4 × 10⁶ cells were used for chromatin purification and subsequent respective immunoprecipitation with either Histone H3 (D2B12) XP^® Rabbit mAb (CST, positive control) or ELK1 antibody (ab32106, Abcam)). The enrichment of the DNA sequences was analyzed by RT-qPCR using GoTag®qPCR Master Mix (Promega) in the LightCycler^® 96 system (Roche). The SimpleChIP^® Human RPL30 Exon 3 Primers (#7014, CST) were used as positive control. The following primer pair was used for the detection of the respective Rad51 promotor sequence: forward: 5′-TCTTCTCGAGCTTCCTCAGC-3′, reverse: 5′-AGCGCTCTTGTGGTTTGTTT-3′). The detected signals were normalized using the percent input method (%input = 100 ×  $2^{\left({\mathrm{Ct}}_{100\%\mathrm{input}}-{\mathrm{Ct}}_{\mathrm{IP}}\right)}$ ; Ct_100%input = Ct_input − log₂ (dilution factor)).

Alisol F and 25-anhydroalisol F were isolated from A. orientale by one of the authors (Q. Ma) using our previously-established method and provided with a purity of 98.0% determined by high-pressure liquid chromatography. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin, and penicillin-streptomycin-amphotericin (PSA) were purchased from Gibco BRL Co. Ltd. (Gaithersbug, MD, USA). Lipopolysaccharide (LPS) from Escherichia coli 055:B5, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dexamethasone (DXM), d-galactosamine (d-gal) and Trizol reagent were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The Griess reagent, the protein extraction kit and BCA protein assay kit were obtained from Beyotime Institute of Biotechnology (Beijing, China). Go Tag^® qPCR Master Mix and GoScript^TM Reverse Transcription System were purchased from Promega (Madison, WI, USA). Rabbit polyclonal antibodies against inducible nitric oxide synthase (iNOS), COX-2, p-p38 (Thr180/Tyr182), p38, p-ERK1/2 (Thr202/Try204), ERK1/2, p-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, p-p65 (Ser536), p65, GAPDH, p-IκB-α (Ser32), IκB-α, p-STAT3 (Tyr705), STAT3, goat-IgG HRP, and lamin B1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse TNF-α, IL-1β, and IL-6 ELISA kit were from R and D systems (Abingdon, UK).

Total RNA was isolated from the non-irradiated control cells and UV-and/or IR-irradiated cells using the RNeasy MiniKit (Geneaid Biotech Ltd., Taipei, Taiwan) according to the manufacturer. Reverse transcriptase reaction was performed with 2 µg of total RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA) according to the manufacture’s recommendation. Quantitative PCR was performed using ABI 7500 Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA). PCR was performed using the GoTag qPCR Master Mix (Promega). The following thermocycling conditions were used: incubation at 95 °C for 2 min, followed by 40 cycles of amplification at 95 °C for 15 s, and then 60 °C for 60 s. The threshold cycle is defined as the cycle number at which the fluorescence corresponding to the amplified PCR product was detected. The PCR arbitrary units of each gene were defined as the mRNA levels normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in each sample. The following primer sequences were used: GAPDH (forward primer) 5′-GAAATCCCATCACCATCTTCCAGG-3′, GAPDH (reverse primer) 5′-GAGCCCCAGCCTTCTCCATG-3′, TNF-α (forward primer) 5′-AGCCCATGTTGTAGCAAACC-3′, TNF-α (reverse primer) 5′-TGAGGTACAGGCCCTCTGAT-3′, IL-6 (forward primer) 5′-CACAGACAGCCACTCACCTC-3′, IL-6 (reverse primer) 5′-TTTTCTGCCAGTGCCTCTTT-3′.

Genomic DNA was extracted from whole blood or cardiac biopsies using standard procedures. Quantitative real-time PCR was carried out using GoTag qPCR Master Mix (Promega) on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions and with normalization to the RPPH1 gene. Primer sequences are available on request. As a reference, genomic DNA from the HapMap individual NA10851 was obtained from the Coriell Cell Repositories (New Jersey, USA).

Total RNAs were isolated from cells using the RNeasy minikit (QIAGEN, Leiden, The Netherlands), and reverse transcribed into cDNAs using M-MLV reverse transcriptase (Promega, Madison, WI, USA). qPCR was conducted to detect the levels of MCL1, Sp1, and SIRT3 mRNA using GoTag qPCR Master mix (Promega, Madison, WI, USA). The primer sequences used are provided in Supplementary Table S1. To measure SIRT3 mRNA stability, the transcription of ABZ- or ABZ plus SB202190-treated cells was inhibited by incubation of actinomycin D (10 μg/mL) for 0.5, 1 and 2 h.