Sybr green

Malonic acid (MA), 2′, 7′-dichlorodihydrofluorescein diacetate (DCF-DA), dimethyl sulfoxide, and epigallocatechin-3-gallate (EGCG), as a positive control of ROS induction, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and trypsin-ethylenediaminetetraacetic acid were purchased from Welgene (Gyeongsan, Korea). SYBR green was purchased from GeneAll (Seoul, Korea). All antibodies (anti-heme oxygenase 1 (HO-1), anti-superoxide dismutase 1 (Sod1), anti-nuclear factor-erythroid 2-related factor-2 (Nrf2), anti-p-inhibitor of NF-κB (IκB), anti-IκB, anti-p-p65, anti-p65, anti-p50, anti-Cox-2, anti-IL-6, anti-TNF-α, anti-p-JNK, anti-JNK, anti-p-ERK, anti-ERK, anti-p-p38, anti-p38, anti-p-c-Jun, anti-c-Jun, anti-c-Fos, anti-p-Smad2/3, anti-Smad2/3, anti-collagen type I alpha 1 (Col1a1), anti-Col3a1, and anti-glyceraldehyde 3-phosphate dehydrogenase (Gapdh)) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

In this experiment, the nSe-associated variations in the expressions of target genes were addressed in leaves (before RNA extraction, leaves were kept in −80 °C). RNA was purified from leaves that were well-grounded in liquid nitrogen using DEPC Water (Bio Basic, Canada), triazole (GeneAll Biotechnology Co, South Korea), and Dnase I (Fermentase, USA). Then, the accuracy of RNA extraction was verified using a Nanodrop (Thermo Scientific^™NanoDrop Model 2000c). Next, the synthesis of complementary DNA (cDNA) was carried out using a thermocycler (PEQLAB, 96Grad). In Table 2, the designed forward and reverse sequences of primers for Phenylalanine Ammonia Lyase (McPAL)- (XM_022284778), 4-Coumaroyl CoA ligase (Mc4CL)- XM_022281848, transcription Factor WRKY1 (NW_019104495), and Elongation factor (a housekeeping gene) are depicted. After that, the transcription rates of the target genes were estimated according to common SYBR green (GeneAll, South Korea) and the real-time quantitative PCR procedure (Applied Biosystems StepOne^™ Real-Time PCR). The delta CT protocol was utilized to calculate the expression rates presented as a fold difference.