Measuring ALKBH7 Knockdown Effects on Mitochondrial Energy

ALKBH7-knockdown effects on mitochondrial energy activity^{41 (link)} were measured using a Seahorse Bioscience XF96 analyser (Seahorse Bioscience Inc.) and the Seahorse XFp Cell Energy Phenotype Test Kit (Agilent) according to the manufacturer’s instructions. Both siControl and siALKBH7 HepG2 cells were seeded at 2 × 10⁴ cells per well with 7 replicates in Seahorse XF96 cell culture microplates (Agilent). Cells were grown overnight and incubated with XFp medium for around 1 h in a non-CO₂ incubator before initiating the cell energy phenotype assay. The 96-well sensor cartridge was hydrated in 200 μl XF calibrant solution (Seahorse Bioscience Inc.) overnight at 37 °C before plate calibration. For the cell energy phenotype assay, cells were treated with 1 μM oligomycin followed by three serial injections of FCCP at final concentrations of 0.5, 1.0 and 2.0 μM. Seahorse Wave software (v2.6) was used for data processing, including detection of outliers and cell number normalization. The final reports for the oxygen consumption rate and extracellular acidification rate signals were generated using the Seahorse XF Cell Energy Phenotype report generator.