Stepone v2

HEK293T cells were seeded into 75 cm² flasks at a density of 5 × 10⁶ cells per flask and cultivated for 12–20 h. Cells were then co-transfected with two helper plasmids (pHelper and pAAV-RC; Stratagene) and one of the pAAV plasmids (pAAV-MCS, pAAV-DCUg-TER, pAAV-DCUg-EZH2, pAAV-DCUg-RelA, and pAAV-DCUg-TER) using Lipofectamine 2000 according to the manufacturer’s instructions. Following transfection, cells were cultured with fresh medium for 72 h. Viruses were then collected and purified as previously described.³⁶ (link) Titers of AAVs were determined by qPCR using the primers AAV-F/R (Table S3). A 20 μL qPCR reaction contained 1 × SYBR Green Real-time PCR Master Mix (Roche), 0.25 μM primers, and 2 μL virus or 2-fold dilutions of a standard DNA (a 159-bp AAV genomic DNA (gDNA) fragment pre-amplified with primers AAV-F/R). The qPCR program was performed as follows: 95°C for 10 s, 45 cycles of 95°C for 15 s, and 60°C for 1 min. A melting curve was then constructed to monitor PCR amplification specificity. Data analysis was performed using Applied Biosystems StepOne v2.3. The concentration of the virus genome (vg) was calculated according to the standard curve. Quantified viruses were aliquoted and kept at −80°C for later use. The obtained viruses were referred to as rAAV-MCS, rAAV-NT, rAAV-TERT, rAAV-EZH2, rAAV-RelA, and rAAV-TER.

To determine whether APS, bassorin, and tragacanthin influence MTX resistance in CCRF-CEM cells, the relative mRNA expression levels of MDR1 in the CCRF-CEM cell lines, sensitive and resistant to MTX, were assessed by quantitative real-time polymerase chain reaction (RT-PCR) with SYBR Green (Maxima SYBR Green/ROX qPCR Master Mix (2X), UK) in a Step One Plus RT-PCR machine (Applied biosystem, Faster city, CA, USA). The total volume of the RT-PCR reaction, 10 μL, contained 1.6 μL synthesized cDNA (0.1 μg/μL), 0.4 μL of 10 μM solutions of each forward and reverse primers, 5 μL of SYBR green, and 2.6 μL of ddH₂O. Primer sequences are summarized in Table 1. These primers were adopted from previous studies (5 (link)). The specificity of the primers was verified by Blast analysis at NCBI.
The 2^-ΔΔCt method was used to determine the relative expression ratio between the target gene (MDR1) and the housekeeping gene used as an internal control (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). Data were analyzed with sequence detector software (Step One v2.3, Applied Biosystems, USA).