• Authors: De Backer J. et al.
  • Year: 2021
  • Journal: Sci Rep 11(1)
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell types:
    1. Name: A375
      Description: Human skin melanoma cells
      Known as: A-375
    2. Name: Hep-3B


3 × 10^5 Hep3B or 3.5 × 10^5 A375 cells were transiently transfected with 300 ng reporter plasmid and YFP-HIF-1α or YFP-HIF-2α as indicated, in a 6-well format using JetOptimus. To control for differences in transfection efficiency and extract preparation, 25 ng pRL-SV40 Renilla luciferase reporter vector was co-transfected. Cultures were evenly split onto 12-well plates 24 h after transfection.


Cytoglobin (CYGB) is a ubiquitously expressed protein with a protective role against oxidative stress, fibrosis and tumor growth, shown to be transcriptionally regulated under hypoxic conditions. Hypoxia-inducible CYGB expression is observed in several cancer cell lines and particularly in various melanoma-derived cell lines. However, reliable detection of hypoxia-inducible mRNA levels by qPCR depends on the critical choice of suitable reference genes for accurate normalization. Limited evidence exists to support selection of the commonly used reference genes in hypoxic models of melanoma. This study aimed to select the optimal reference genes to study CYGB expression levels in melanoma cell lines exposed to hypoxic conditions (0.2% O2) and to the HIF prolyl hydroxylase inhibitor roxadustat (FG-4592). The expression levels of candidate genes were assessed by qPCR and the stability of genes was evaluated using the geNorm and NormFinder algorithms. Our results display that B2M and YWHAZ represent the most optimal reference genes to reliably quantify hypoxia-inducible CYGB expression in melanoma cell lines. We further validate hypoxia-inducible CYGB expression on protein level and by using CYGB promoter-driven luciferase reporter assays in melanoma cell lines.