L02 cells at a density of 1 × 106 cells/well were seeded in six-well plates and cultured overnight. After incubation with NOB/SD or Free NOB at NOB concentrations of 12 μM for 24 h, the treated L02 cells were exposed with 7.5 mM APAP for another 24 h. Total RNA was extracted using TRIzol (Thermo, Massachusetts, USA) and converted into cDNA using a reverse transcription kit (Vazyme, Nanjing, China) according to the manufacturer’s protocols. The RNA expression levels of HO-1 and NQO1 were analyzed using quantitative real-time PCR system (Bio-Rad, USA).25 (link) The primers in the reaction are shown in Table 2 .
The Primer Sequences for qPCR to Assess the Expression of HO-1, NQO1 and GAPDH in L02 Cells
| Target | Primer | Sequence |
|---|---|---|
| GAPDH | FP | 5′-CTGCACCACCAACTGCTTAG-3′ |
| RP | 5′-AGGTCCACCACTGACACGTT-3′ | |
| HO-1 | FP | 5′-TGCGGTGCAGCTCTTCTG-3′ |
| RP | 5′-GCAACCCGACAGCATGC-3′ | |
| NQO-1 | FP | 5′-TGGCTAGGTATCATTCAACTC-3′ |
| RP | 5′-CCTTAGGGCAGGTAGATTCAG-3′ |
