Apocynin

Green GS cells were derived from C57BL6/Tg14(act-EGFP-OsbY01) transgenic mice on a DBA/2 background (Kanatsu-Shinohara et al. 2003 (link)). We also derived GS cells from 7- to 10-d-old Nox1 KO, B6.129-Hif1a^tm3Rsjo/J, or Cdkn1a KO mice, as described previously (Kanatsu-Shinohara et al. 2014b (link)). Myc DKO GS cells have been described (Kanatsu-Shinohara et al. 2016 (link)). For hypoxic culture, GS cells were placed into plastic bags and gassed with 1% O₂/5% CO₂/94% N₂. When cells were cultured under 5% or 10% O₂, the CO₂ level was maintained at 5%, but the N₂ level was reduced to 90% or 85%, respectively. Where indicated, GS cells were cultured with 30 µM hydrogen peroxide (Wako), 100 µM MitoTempo, 2 mM LPA (both from Sigma), and 1 mM apocynin (Tokyo Chemical Industry).
For Cdkn1a and KD, p21-shRNA-1, p21-shRNA-2, and empty control vectors were used (Dr. Sally Temple, Albany Medical College, NY) (Fasano et al. 2007 (link)). A mixture of lentiviral particles was used for transfection. For cDNA overexpression, mouse Mycn cDNA was inserted into the CSII-Ef1a-IRES-Venus vector. Virus particles were prepared as described previously (Shinohara and Kanatsu-Shinohara 2020 (link)). The multiplicity of infection was adjusted to 10.0. AxCANCre was used for CRE-mediated deletion of target genes in GS cells (MOI = 2.0).