M mlv assay kit

Cells that underwent the indicated treatments were harvested for RNA isolation using the Trizol reagent (Invitrogen, USA) according to manufacturer's instructions, and the RNA concentration was measured using a UV spectrophotometer. One microgram of total RNA was used as template for reverse transcription in accordance with the instructions of the M-MLV assay kit (Invitrogen). The primer sequences used in this study are shown in Table S1. PCR reactions (10 μl) were performed in a Bio-Rad PCR amplifier. The real-time PCR reaction system included 5 μl of SYBR Premix Ex Taq (Thermo Fisher, USA), 0.5 μl of forward primer (10 pmol/μl), 0.5 μl of reverse primer (10 pmol/μl), 1 μl of cDNA template (500 ng/μl), and 3 μl of ultrapure H₂O.
The amplification conditions comprised 40 cycles at 95 °C for 5 s and 65 °C for 30 s. The results were analyzed by the Bio-Rad CFX Manager software.

Total cellular RNA was isolated from cultured cells using Trizol reagent (Invitrogen) and reverse transcribed into cDNA using the M‐MLV assay kit (Invitrogen) according to the manufacturer's instructions. These cDNA samples were subjected to qPCR amplification of different genes with their primer sequences (Table S1) in 10 µL of the qPCR mixture in a Bio‐Rad PCR amplifier (Hercules, CA). The qPCR mixture included 5 μL SYBR Premix Ex Taq (Vazyme, Nanjing, China), 0.5 μL of each primer (10 pmol/μL), 1 μL cDNA template and 3 μL ddH₂O. Amplification conditions were initially set at 95°C for 5 minutes and then 40 cycles of 95°C for 5 seconds and 65°C for 30 seconds. The results were analysed using the Applied Biosystems QuantStudio 7 Flex software using 2^−(Ct‐Cc) (Ct and Cc were the mean threshold cycle differences after normalizing to β‐actin).

Trizol reagent (Invitrogen) was used to isolate total RNA. According to the instruction of the M-MLV assay kit (Invitrogen), RNA was reverse-transcribed into cDNA. The primer sequences were as follows: AGR2-Forward: 5′-TTGTCCTCCTCAATCTGGTT-3′; AGR2-Reverse: 5′-ATCGGCTCTAACTGTCAGAGA-3′; GAPDH-Forward: 5′-GGGAAACTGTGGCGTGAT-3′; GAPDH-Reverse: 5′-GAGTGGGTGTCGCTGTTGA-3′. SYBR Green qPCR SuperMix for quantitative PCR was purchased from Vazyme Company. Quantitative PCR was performed using the ABI PRISM^® 7500 Sequence Detection System. Amplification conditions were set to 40 cycles at 95°C for 5 s and 65°C for 30 s.