Transzol up reagent

RAW264.7 cells (1 × 10⁶ cells/well) were treated with the same way of ELISA and lysed in TransZol Up reagent (TransGen Biotech, Beijing, China). RNA was extracted according to the instructions from the manufacturer and quantified with a microplate reader (Tecan Infinite 200 Pro, Switzerland). cDNA was synthesized with Primescript™ First-Strand cDNA Synthesis kit (Takara, Beijing, China). The real-time polymerase chain reaction (PCR) was performed using qTOWER 3G (Analytic Jena, Jena, Germany) with TB Green® Premix Ex Taq™ II kit (Takara, Beijing, China). The obtained amplification data were calculated using GAPDH as internal reference. The specific primers are shown in Table 1.

H1299 cells were cultured in 12-well plated at 2.5 × 10⁵ cell/well in the presence of compound 15 (0.5, 1, and 2 μM) for 24 h. Total RNAs present in the cultured cells were extracted using the TransZol™ Up Reagent (TransGen Biotech, Beijing, China). Gene expression was detected via quantitative real-time PCR (qRT-PCR) and SYBR® Premix EX Taq™ II (TaKaRa Bio, Otsu, Japan) was used to perform the analysis.

Plant samples collected at the respective time points were ground to powder in a pre-chilled sterilized mortar and pestle with sufficient liquid nitrogen, and were used to isolate total RNA using the TransZol Up reagent (Transgen, Beijing, China) according to the manufacturer’s instructions. The concentration and quality of isolated RNA were determined by a NanoDrop ND-1000 spectrophotometer (Thermo-Fischer Scientific, Waltham, MA, USA) and by 1% (w/v) agarose gel electrophoresis. First-strand cDNA was synthesized from isolated RNA by using a Transcript one-step gDNA removal and cDNA synthesis supermix kit (Transgen) as per the manufacturer’s instructions.

Total RNA was extracted from cells with TransZol UP reagent (Transgene, Strasbourg, France) and reverse transcribed with the Revert Aid First Strand cDNA Synthesis Kit (Transgene). The following q-PCR primer sequences were used: LHPP forward: 5-CAAACTGTGTGGTAATTGCAGA-3, reverse: 5-CCAGAGGTCTCCTTGTAGTAAC-3; snail forward: 5-CCTTCGTCCTTCTCCTCTACTT-3, reverse: 5-GCTTCGGATGTGCATCTTGA-3; laminin-5 forward: 5-CCATGAATTTCTCCTACTCGCC-3, reverse: 5-CTCCGATGCTGATATCCTTGAT-3; L1CAM forward: 5-GCTGGTTCATCGGCTTTGTG-3, reverse: 5-CTGTACTCGCCGAAGGTCTC-3; N-cadherin forward: 5-CGATAAGGATCAACCCCATACA-3, reverse: 5-TTCAAAGTCGATTGGTTTGACC-3; vimentin forward: 5-AGTCCACTGAGTACCGGAGAC-3, reverse: 5-CATTTCACGCATCTGGCGTTC-3; E-cadherin forward: 5-AGTCACTGACACCAACGATAAT-3, reverse: 5-ATCGTTGTTCACTGGATTTGTG-3;GAPDH forward: 5-CACCCACTCCTCCACCTTTGA-3, reverse: 5-TCTCTCTTCCTCTTGTGCTCTTGC-3. PCR was performed with Fast Start Universal SYBR Green Master Mix (Roche, Basel, Switzerland), and the fluorescence was measured using an ABI 7500 Real Time System (Applied Biosystems, Life Technologies, Foster City, CA, U.S.A.) by following the manufacturer’s instructions. Data were analyzed using the 2^−ΔΔCt method, and GAPDH was regarded as an internal control.

Total RNA was isolated using TransZol Up reagent (TransGen Biotech, Beijing) according to the manufacturer. Complementary DNA was synthesized using AccuPower^® RT PreMix (Bioneer, Daejeon, Republic of Korea), and products of interest were amplified by qPCR using SYBR green and the CFX Connect Real-Time System (Bio-Rad Laboratories Inc., Hercules, CA, USA), as described in the previous study [24 (link)]. Table 2 shows the specific primers used in this study.

Total RNA was extracted with TransZol Up reagent (TransGen Biotech), followed by cDNA synthesis with TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech). Quantitative real-time PCR (qRT-PCR) was then performed using PerfectStart Green qPCR SuperMix (TransGen Biotech) on an ABI QuantStudio 5 instrument. Gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase. The sequences of the primers used are listed in Supplementary file 2: Table S2.