Figure 1
Transformation of human MSC leads to an increase in intracellular ROS that contributes to the transformed phenotype. (A) Schematic diagram of MSC step-wise transformation. Cell lines were named according to the number of oncogenic 'hits’ introduced by sequential retroviral transduction. MSC0 are primary MSC, MSC1 are MSC0 expressing hTERT, MSC3 are MSC1 expressing E6 and E7 from HPV-16, MSC4 are MSC3 expressing ST antigen from SV40, and MSC5 are transformed MSC (tMSC) obtained after expression of H-RasV12 in MSC4. (B) Intracellular ROS concentration measured by flow cytometry after staining with MitoSOX Red. ROS amounts are presented as the geometrical mean fluorescence intensity (± SD) from at least three experiments. (C) Intracellular ROS concentration of MSC expressing different oncogenes measured by flow cytometry after staining with CM-H2DCFDA. ROS amounts are presented as the geometrical mean fluorescence intensity (± SD) from at least three experiments. (D) ROS production in tMSC measured by CM-H2DCFDA staining after 4 hours treatment with 2.5 mM N-acetyl-L-cysteine (NAC) or 50 μM ascorbic acid (AsA). ROS levels produced by MSC3 are also shown. (E) Cell viability after 6 and 24 hours treatment with 2.5 mM NAC or vehicle control was addressed by using CellTiter AQueousOne Solution Cell Proliferation Assay (Promega) following the manufacturer’s instructions. A representation of three different experiments is shown and data were calculated from the average of four replicate wells. Results are shown as percentage of viable cells referred to control cells (MSC3) treated with vehicle control. (F) Soft agarose transformation assay of untreated and 2.5 mM NAC treated tMSC. Colonies were photographed at x40 magnification (top) and counted in triplicate wells (bottom) after 12 days in culture. P values are *P < 0.05, **P < 0.005 and ***P < 0.0005.