MMAB small hairpin RNA (shRNA) transfection and function analysis

MMAB shRNA transfection was performed according to the previous report [28]. Briefly, the shRNA specific for MMAB (shMMAB) was designed according to Genebank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_052845","term_id":"1519246003","term_text":"NM_052845"}}NM_052845 (Uniprotkb number of MMAB_HUMAN) wherein the hairpin RNA sequence is GGAGGCTCACTTAAAGTAT. The Interference vector GV248 was purchased from Shanghai Genechem Company (Shanghai, China). Huh-7 cells were transfected with MMAB-shRNA or its non-targeting control shRNA at the concentration of 0.8 or 2 μg per well (24 or 6 well-plate array) for 6 h in serum-free culture medium. Fetal bovine serum was then added to a final concentration of 10%. 48 h later, the cells were treated with EFV or H₂O₂ or Rotenone, and used for ROS analysis, treated with EFV, or lysed and used for WB detection and real-time RT-PCR to verify the effect of shMMAB, expression of ALDH3A2 or for amino acid analysis. For amino acid analysis, the cells were disrupted through freezing and thawing three times, followed by grinding in liquid nitrogen. Then the broken cells were centrifuged at 12000 rpm at 4°C for 15 min, and the supernatant was collected. An equal volume of 10% Trichloroacetic acid was added to the above supernatant. After 60 min at 4°C, the sample was centrifuged at 15000 rpm at 4°C for 30 min. The supernatant was withdrawn and adjusted to pH 1.7 to 2.2 using 8 mol/L NaOH (usually 24 μL 8 mol/L NaOH for 800 μL samples), then centrifuged at 15000 rpm at 4°C for 15 min and filtered using 0.45 μg filter membrane. The filtrate (with volume more than 200 μL) was freeze-dried and re-dissolved with 400μL hydrochloric acid (0.02 mol/L). After centrifugation at 15000 rpm at 4°C for 15 min, the supernatant was withdrawn. 50 μL was injected into an amino acid analyzer (Hitachi L8500, Tokyo, Japan). For real-time RT-PCR analysis of ALDH3A2, the sense and antisense primers are ACAATACCCAGGAAAGTG, and GAAGGTGCTAACAAACTCA, respectively.