Figure 1. H19 RNA is largely induced in response to hypoxic stress and moderately by hypoxia-mimicking condition triggered by CoCl₂ in Hep3B.

Hep3B cells were cultured under normal culture conditions for 24 hours before hypoxic or CoCl₂ manipulation. Cells were either placed into an aneoropack rectangular jar to create a hypoxic condition within an hour, or left under normal culture conditions. Incubation lasted for 24 hours before RNA extraction. (A) Shown are RT-PCR products of H19 gene (28 PCR cycles) cultured under normal conditions-lane 1, or hypoxic conditions-lane 2 (lane M indicates the marker, and C is a PCR blank without a target). (B) PCR for Histone H3.3 as a positive control for RT-PCR integrity. Shown also are RT-PCR products of both the H19 gene (32 PCR cycles) (C), the GAPDH gene (D) for untreated Hep3B (lane1) and for 50, 100, 200, 300 and 400 µM CoCl₂ treated cells (lanes 2, 3, 4, 5 and 6, respectively). Incubation with the indicated concentrations of CoCl₂ lasted for an additional 22 hours before RNA extraction. QPCR analysis for H19 RNA levels normalized to β-actin in Hep3B treated CoCl₂ is shown in (E) where the numbers above the bars indicate the concentrations of CoCl₂ used.