Total rna extraction kit

Total DNAs were extracted from the terminal buds of different samples according to the cetyl trimethylammonium bromide (Beijing Chemical Co., Beijing, China) method, as described by Zhang et al. [23] , RNase (Invitrogen, Carlsbad, CA, USA) was used to render the DNA free of genomic RNA contamination. Total RNAs were extracted following by an Aidlab total RNA extraction kit (Aidlab, Beijing, China). RNase free - DNase I (Invitrogen) was used to render the RNA free of genomic DNA contamination. The DNAs and RNAs were assessed with NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA).

TSG (purity = 94.7%) was obtained from the National Institutes for Food and Drug Control (Beijing, China). Fetal bovine serum (FBS) and alpha modification of Eagle’s minimum essential medium (α-MEM) medium were purchased from Hyclone (Logan, UT, USA). Methyl thiazolyl tetrazolium (MTT) and LY-294002 were purchased from Sigma Aldrich (St. Louis, MO, USA). The total RNA extraction kit was purchased from Aidlab (Beijing, China). The cell cycle analysis kit and materials for Western blot were purchased from Beyotime Biotechnology (Nantong, Jiangsu, China). Reverse transcriptase and qPCR Mix were purchased from TransGen Biotech (Beijing, China). Akt1 antibody was purchased from Signalway Antibody (College Park, MD, USA). PAkt1 (ser473) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). P-PI3K (p85) antibody was purchased from EnoGene (Nanjing, China). PI3K (p85), M-CSF, Osx, and Col1a1 antibodies were purchased from Wanleibio (Shenyang, China). The Runx2 antibody was purchased from Cusabio (Wuhan, China). GAPDH, β-actin, and β-tublin antibodies were purchased from Abclonal (Wuhan, China). OPG and RANKL ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

RIMVECs were seeded in a 6-well cell culture plate at a concentration of 1 × 10⁵ cells/well in 2 mL DMEM per well. When the cells reached 90% confluency, they were divided into control group, model group (LPS+ATP), and quercetin groups at the doses described in the paragraph of the in vitro experiment and incubated for 12 h. Total RNA Extraction kit and reverse transcriptase were purchased from Aidlab (Beijing, China). Reverse transcription was performed using the Prime Script RT reagent kit (TAKARA). Quantitative reverse transcription PCR (RT-qPCR) was performed using a CFX9600 apparatus (Bio-Rad, Hercules, CA, USA). The RT-qPCR conditions were as follows: pre-denaturation at 94°C for 30 s; 40 cycles of denaturation at 94°C for 5 s, annealing at 60°C for 15 s. The 20 μL reaction mixture was composed of 0.4 μL (10 μmol/L) forward primer, 0.4 μL (10 μmol/L) reverse primers, 0.8 μL cDNA, 8.4 μL dd.H₂O, and 10 μL qPCR SuperMix. The gene sequences of rat-derived TLR4, GSDMD, caspase-1, IL-1β, and IL-18 genes were confirmed in GenBank. Primer Premier 5.0 software was used to design the primers, which were then synthesized by Sangon (Shanghai, China). The target gene expression was calculated by normalizing it with the expression of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2^−ΔΔCt method. The specific primer sequences of the target genes are listed in Table 1.

Total RNA was extracted from A. sinensis calli and tissues using a Total RNA extraction kit (Aidlab, China), supplemented with on-column DNA digestion per the manufacturer’s instructions. Single-stranded cDNA was synthesized from the total RNA using a PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time; Takara, Japan) per the manufacturer’s protocols and subjected to qRT-PCR analysis using the LightCycler (Roche Diagnostics, Indianapolis, IN, USA). The relative expression level for each candidate genes under MeJA treatment was calculated using the 2^–ΔΔCq method and the 2^–ΔCq method for the tissue expression profile. GADPH was used as the internal reference gene. The primers used for qRT-PCR reactions were designed for the selected bHLH genes using Primer Premier 5.0 and listed in the Supplementary Table S4.

True leaves, cotyledons, seed roots, secondary roots and stems were sampled from cotton plants at the second true-leaf stage with three biological repetitions. All the samples were immediately frozen in liquid nitrogen and stored at − 80 °C. RNA of each sample was extracted using a Total RNA Extraction kit (Aidlab, Beijing, China). First strand cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotec Co. Ltd.) following the manufactures protocol. Primers for qRT-PCR were designed with the Primer 5.0 software. All primers used in the experiment are listed in Additional file 7: Table S4 and cotton UBQ7 was used as an internal control. The amplification reactions of qRT-PCR were performed with Lightcycler 96 system (Roche) using SYBR the premix Ex taq (TakaRa) with the following parameters: 30 s initializing denaturation at 95 °C; followed by 45 cycles of 10 s denaturation at 95 °C, 10 s annealing at 54 °C, and 20 s extension at 72 °C. In addition, the default setting for the melting curve stage was chosen. The relative expression levels were calculated by the method of 2^-ΔΔCt. The heatmap for expression profiles was generated with the Mev 4.0 software.

LPS (Escherichia coli O55:B5) and glycine were products of Sigma (St. Louis, MO, USA). Alcian blue (pH 2.5) staining kit was obtained from Vector Laboratory (Burlingame, USA). Commercial mouse inflammation panel kit was purchased from BioLegend (San Diego, USA). Primers were synthesized by Sangon Biotech Co. (Shanghai, China). Total RNA extraction kit was purchased from Aidlab Biotechnologies (Beijing, China). FastQuant RT Kit (with gDNase) and SuperReal PreMix Plus (SYBR Green) were purchased from TIANGEN Biotech (Beijing, China). Antibodies against GAPDH, Actin, phosphor-P65 (p-P65), NLRP3, Procaspase1, Cleaved caspase1, NRF2, and Beclin1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against P65, P62, autophagy related gene 5 (ATG5), and microtubule-associated protein light chain 3 (LC3) were products of Cell Signaling Technology (Danvers, MA, USA). Antibodies against TLR4, MYD88, ASC, HO1, NQO1, GSTA4, HSP40, HSP70, and HRP-conjugated rabbit anti-mouse, and mouse anti-rabbit secondary antibodies were gained from Sangon Biotech Co. (Shanghai, China).