Cdna master mix

Total RNA was extracted from YCC-2 and SNU-668 cells treated with G. thunbergii using RNAiso Plus reagent (TaKaRa Bio, Japan), and cDNA was synthesized using cDNA Master Mix (ToYoBo, Japan) according to the manufacturer’s protocol. Reverse transcription PCR was performed as described previously (Kim et al. 2018 (link)) using the following PCR primer sequences: Bcl-2, 5ʹ-GGATGCCTTTGTGGAAAACCCTGT-3ʹ (forward) and 5ʹ-AGCCTGCAGCTTTGTTTCAT-3ʹ (reverse); Bax, 5ʹ-TTTGCTTCAGGGTTTCATCC-3ʹ (forward) and 5ʹ-CAGTTGAAGTTGCCGTCAGA-3ʹ (reverse); caspase-3, 5ʹ-TTTTTCAGAGGGGATCGTTG-3ʹ (forward) and 5ʹ-CGGCCTCCACTGGTATTTTA-3ʹ (reverse); PARP, 5ʹ-TGGAACATCAAGGACGAGCT-3ʹ (forward) and 5ʹ-GCATCGCTCTTGAAGACCAG-3ʹ (reverse); GAPDH, 5ʹ-GGCTGCTTTTAACTCTGGTA-3ʹ (forward) and 5ʹ-ACTTGATTTTGGAGGGATCT-3ʹ (reverse). PCR products were used for 1% agarose gel electrophoresis with RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology).

The BV2 microglia cells were incubated for 24 h with LPS alone or after 1 h of pre-treatment with malonic acid. Total RNA was isolated using RNAiso Plus reagent (TaKaRa Bio, Japan), and cDNA was synthesized using cDNA MasterMix (ToYoBo, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR was conducted using the AMPIGENE® qPCR Green Mix Hi-ROX (Enzo, USA) according to the manufacturer’s instructions. The mRNA expression of iNOS, IL-1β, IL-6, and TNF-α was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used in this study were as follows: iNOS, 5ʹ-CGGGTTGAAGTGGTATGCAC-3ʹ (forward) and 5ʹ-GCTGTGTGGTGGTCC ATGAT-3ʹ (reverse); IL-1β, 5ʹ-GTGTCTTTCCCGTGGACCTT-3ʹ (forward) and 5ʹ-TCGTT GCTTGGTTCTCCTTG-3ʹ (reverse); IL-6, 5ʹ-CCTTCCTACCCCAATTTCCA-3ʹ (forward) and 5ʹ-CGCACTAGGTTTGCCGAGTA-3ʹ (reverse); TNF-α, 5ʹ-GGCCTCTCTACCT TGTTGCC-3ʹ (forward) and 5ʹ-TAGGCGATTACAGTCACGGC-3ʹ (reverse); GAPDH, 5ʹ-TGCACCACCAACTGCTTAG-3ʹ (forward) and 5ʹ-GGATGCAGGGATGATGTTC-3ʹ (reverse).

Total RNA was isolated using RNAiso Plus reagent (TaKaRa Bio, Kusatsu, Japan), and cDNA was synthesized using cDNA Master Mix (ToYoBo, Ōsaka, Japan), according to the manufacturer’s instructions. Then, PCR was performed by using HiPi Plus 5 × PCR Master Mix (Elpis Biotech, Daejeon, Korea). The primer sequences used in this study were as follows: LAMB1, 5′-AGGTTGGAGCTGCCTCAGTA-3′ (forward) and 5′-ACACTCCCTGGAAACAGTGG-3′ (reverse); JUN, 5′-CCCCAAGATCCTGAAACAGA-3′ (forward) and 5′-CCGTTGCTGGACTGGATTAT-3′ (reverse); CEBPB, 5′-CAAGAAGCCGGCCGAGTAC-3′ (forward) and 5′-TTGTCCACGGTCTTCTTGGC-3′ (reverse); RXRA, 5′-CCTTTCTCGGTCATCAGCTC-3′ (forward) and 5′-TGTCAATCAGGCAGTCCTTG-3′ (reverse); YY1, 5′-GGATAACTCGGCCATGAGAA-3′ (forward) and 5′-GGTTGTTTTTGGCCTTAGCA-3′ (reverse); GAPDH, 5′-TGCACCACCAACTGCTTAG-3′ (forward) and 5′-GGATGCAGGGATGATGTTC-3′ (reverse). PCR products were separated using 1% agarose gel electrophoresis and visualized using RedSafe nucleic acid staining solution (iNtRON Biotechnology, seongnam, Korea).