Trizol

A total of 10 mg of plant tissue was used for RNA isolation. Total RNA was extracted using the Trizol method (Trizol was purchased from TaKaRa, Dalian) and then treated with DNAse I for digestion using the RNAse-free kit (TaKaRa, Dalian) to decrease potential DNA contamination. The quality of the RNA samples was verified by 1.0% agarose gel electrophoresis (AGE). The RNA concentration and purity were determined with a NanoDrop ND-2000 spectrophotometer. RNA samples with a 260/230 ratio higher than 2.0 and 260/280 ratio between 1.8 and 2.1, as well as 18S and 28S ribosomal RNA bands with a concentration ratio of approximately 1:2, were selected and used in the subsequent experiment. First-strand cDNA was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Dalian) according to the manufacturer’s instructions. The final cDNA products were diluted for RT-qPCR experiments.

Mouse ear tissues were homogenized in TRIzol (Takara) using Beadbeater (Biospec), whereas cells were directly resuspended in TRIzol (Takara). Total RNAs were isolated and reverse transcribed into complementary DNA by using RevertAid First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. RT–qPCR was performed in triplicate using SYBR green master mix (Roche) on a StepOnePlus Real-Time PCR System (Applied Biosystem). Samples with a low yield of RNA were predetermined and excluded. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the comparative ∆∆C_T method was used to determine the quantification of gene expression. Primers used in this paper are listed in Supplementary Table 3.

The HCC and adjacent normal tissue samples were retrieved from liquid nitrogen and grinded to tiny particles, before incubation with TRIzol (Takara, Otsu, Shiga, Japan) to isolated mRNA. HepG2 cells that were harvested in 35 mm dishes were also incubated with TRIzol (Takara) to extract mRNA. 500 ng RNA was applied to synthesis first-strand complementary DNA using first-strand cDNA Synthesis Kit (Takara, Otsu, Shiga, Japan). RT-PCR was performed in a mixture composed of SYBR Green (Takara), 1 μl (0.2 μmol/l) of each primer, and 1.6 μl of complementary DNA from RT-PCR samples. The primer sequences were as follows: AEG-1, 5'-CGGTACCCCGGCTG GGTGAT-3' (forward) and 5'-CTCCTCCG CTTTTTGCGGGC-3' (reverse); MDR-1, 5'-GCCGGGAGCAGTC ATCTGTGG-3' (forward) and 5'-ATCCAT TCCGACCTC GCGCT-3' (reverse); GAP DH, 5'-CAAT GACCCCTTCATTGACC-3' (forward) and 5'-GACAAGCTTCCCGTTC TCAG-3' (reverse). The mRNA levels of AEG-1 and MDR-1 were quantified by the Ct values, and GAPDH was set as an internal control.
Experiments were performed in triplicate. The relative expression levels were evaluated using the ^-ΔΔCt method as described previously (Yang et al., 2014[29 (link)]). Each individual experiment was performed three times independently.

To extract total RNA from MSCs, first, MSCs were washed 3 times with phosphate-buffered saline, TRIzol (TaKaRa, Dalian, China) was added to lyse the cells, and extraction was performed according to the manufacturer’s instructions. Specifically, to extract total RNA from MSCs cocultured with monocytes to assess the RNA level of MCP1 and eliminate interference from MCP1 derived from monocytes, a transwell system was used to perform coculture. MSCs (2 × 10⁴) were seeded in the lower chambers, and CD14+ monocytes (1 × 10⁶) were cultured in the upper chambers. Then, the upper chambers containing monocytes and supernatants were removed, MSCs in the lower chambers were washed 3 times with phosphate-buffered saline, and TRIzol (TaKaRa) was added to lyse the cells. Total RNA was reverse transcribed into cDNA with a PrimeScript PT Reagent Kit (TaKaRa). Quantitative real-time PCR was performed using a SYBR Premix Ex Taq Kit (TaKaRa). The PCR procedure was set as follows: 95 °C for 1 min, 40 cycles at 95 °C for 30 s, 58 °C for 20 s, and 72 °C for 30 s, and finally 72 °C for 5 min for full elongation of the products. The results were analyzed using the 2^−ΔΔCt method, and GAPDH was considered the reference gene to calculate relative gene expression. The forward and reverse primers are listed in Additional file 1: Table S1.

Total RNA was isolated using TRIzol (Takara Bio, Beijing, China) according to the manufacturer’s protocol. In short, total RNA was isolated from liver tissue (60mg) using 1mL TRIzol (Takara) to lyse the cells. Next, we took the supernatant, added 0.3mL of chloroform for separation, and centrifuged at 13,800× g for 10 min at 4 °C. After transferring the upper aqueous phase to another EP tube, 400 μL of pre-chilled isopropanol was added to mix and centrifuge. The supernatant in the tube was discarded, and 1 mL of 75% ethanol pre-cooled at −20 °C was added to wash three times. Finally, 50 μL of Rnase-free water was added to dissolve the RNA and stored in a refrigerator at −80 °C. The quality of total RNA was assessed with Nanodrop ND-1000 spectrophotometer (Bio-Rad, USA) and integrity was further examined using 1% agarose gel electrophoresis.

The sorted cells were stored in the TRIzol (TaKaRa Clontech, Japan). Total RNAs were extracted according to the manufacturer's protocol of TakaRa. Template cDNAs were obtained by reverse transcription of total RNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa Clontech, Japan). Amplification was carried out by using SYBR^® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa Clontech, Japan). The mRNA expression level of β-actin was used as internal control. The relative mRNA levels of P2Y_s were calculated as follows [44 (link)]. P2Ys genes were taken for example and the average CT for β-actin was subtracted from the average value for P2Ys to generate Δ for each sample. CT is the cycle number of PCR amplification. Δ = CT× (P2Ys) - CT× (β-actin). Therefore, ΔΔ = Δ(P2Ys value for the samples at day10 or day21)- Δ(P2Ys value for the samples at day0). Finally, the formula 2^−ΔΔ was taken to calculate the relative mRNA level compared with the control. The sequences of the primers for real-time PCR are listed in Table 1.