Super gelred

After two weeks of cultivation in liquid medium without the selection pressure of the herbicide, cells originating from selected resistant colonies were harvested and genomic DNA was extracted using a plant genomic DNA kit (Tiangen, Beijing, China). According to the sequence of vector pSVrPA/CaMVbar, primers bar1F (5′-TCTGCACCATCGTCAACCACTACA-3′), bar1R (5′-TCAAATCTCGGTGACGGGCAGGAC-3′), rpa3F (5′-TCTTGGGCAGAACATACC-3′), and rpa3R (5′-TCCCCCTGAACCTGAAAC-3′) were designed to amplify the specific bar and rt-PA gene fragments by PCR. PCR products were visualized using gel electrophoresis in a 1.2% agarose gel stained with Super GelRed (US Everbright Inc., Suzhou, China). The probe of rt-PA gene for Southern blotting was synthesized using the 804 bp PCR fragment amplified by primers rpa3F/R. Total genomic DNA (about 10 µg) of each sample was digested with endonucleases HindIII and BamHI (New England Biolabs, Ipswich, MA, USA). Probe synthesis and Southern blotting were conducted with a DIG DNA labeling and detection kit (Roche, Mannheim, Germany).

The total RNA of cells was extracted with TRIzol reagent (Cat# 9108, Takara, Dalian, China) according to the manufacturer’s instructions. With the cDNA Synthesis Kit (Cat# CW2020, Cwbio, Taizhou, China), 1 μg RNA was used for reverse transcription into cDNA. A PCR kit (Cat# CW0690, Cwbio, Taizhou, China) was used to perform PCR under the following conditions: 95 °C for 90 s; followed by 30 cycles at 94 °C for 30 s, 56 °C for 20 s, and 72 °C for 40 s; and a final extension at 72 °C for 10 min. Two percent agarose gel electrophoresis was used to separate PCR products which were stained using Super Gelred (US EVERBRIGHT, Suzhou, China, Cat# S2001). The specific bands were examined by Image J 1.51j8 software, and mRNA levels of target genes were normalized against GAPDH levels [11 (link)]. The primer sequences are presented in Table 1.

To measure the RNA levels of the indicated genes, the total intracellular RNA content was extracted from cells using TRIzol reagent (Takara), and the first-strand cDNA was reversed-transcribed by using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific). The cDNA was quantitated by qRT-PCR using the Bestar® SybrGreen qPCR master mix reagent (DBI® Bioscience). The data shown represent the relative abundance of the indicated RNA normalized to that of GAPDH. The nucleic acid stains (Super GelRed, no.: S-2001) was purchased from US Everbright Inc. The primer sequences for HCV and GAPDH were previously described 28 (link). The primer sequences for p38α, mitogen-activated protein kinase kinase 3 (MKK3) and mitogen-activated protein kinase kinase 6 (MKK6) were as follows p38α: 5'-GCCCAAGCCCTTGCACAT-3' (forward) and 5'-TGGTGGCACAAAGCTGAT GAC-3' (reverse); MKK3: 5'-CCCCAGTCCAAAGGAAAATCC-3' (forward) and 5'-TCTACCACCCCATAGGCTCC-3' (reverse); MKK6: 5'-ATGTCTCAGTCG AAAGGCAAGA-3' (forward) and 5'-CACCTCGTCCCAGTTCCATT-3' (reverse). The titers of the HCV viral supernatant were quantified using a Diagnostic Kit for Quantification of Hepatitis C Virus RNA (PCR-Fluorescence Probing) (KHB Company). All qRT-PCR experiments were performed on an ABI 7500 system according to the manufacturer's instructions.