Rna extraction reagent

Total RNA from treated cells was extracted via RNA Extraction Reagent (Yeasen Biotech, Shanghai, China) according to the manufacturer’s instructions. The concentration and quality of RNA were evaluated by spectrophotometric determination at 260/280 nm, and equal amounts of RNA from each sample were used for cDNA reverse-transcription following the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Biomedical Technology, Beijing, China). cDNA was further applied as a template for real-time quantitative polymerase chain reaction (RT-qPCR) following the manufacturer’s instructions (Mastercycler Gradient, Eppendorf, Germany). The primers were listed as follows:
Syntaxin 17 (STX17): 5′-GAGGGTCCGTCAGTCAAGTT-3′ (forward), 5′-CTGACCCTCAGGCATCCAAT-3′ (reverse)
Synaptosome associated protein 29 (Snap29): 5′-CCCTTCCTGCTTCCAAGGTT-3′ (forward), 5′-CCCTGCGTAACACCTCTTGT-3′ (reverse)
Vesicle-associated membrane protein 8 (Vamp8): 5′-GGAAGCCACGTCTGAACACTT-3′ (forward), 5′-GATGGTGCCCGTAGCAAAGA-3′ (reverse).

Total RNA was isolated from MIHA cells using RNA Extraction Reagent (19201ES60; YEASEN, China). Then, the isolated RNA was reversely transcribed into cDNA using a cDNA Synthesis Kit (11119ES60; YEASEN). Subsequently, qPCR was performed by SYBR Green qPCR Mix (11198ES03; YEASEN) in a real-time PCR system (ABI 7300; ABI, USA). The primer sequences are listed below: BMI1-forward: 5′-AGATCGGGGCGAGACAATG-3′, reverse: 5′-TTTTATTCTGCGGGGCTGGG-3′; GAPDH-forward: 5′-GTCTCCTCTGACTTCAACAGCG-3′, reverse: 5′-ACCACCCTGTTGCTGTAGCCAA-3′. The expression values were normalized to GAPDH using the 2^−ΔΔCT method [16 (link)].