Figure 3. MEOX1 and MEOX2 activate transcription of the p16^INK4a gene in HUVECs.

A) Relative level of p16^INK4a mRNA in HUVECs. Total RNA was isolated from HUVECs 72 hours after adenoviral transduction at a multiplicity of infection (MOI) of 250 and the amount of p16^INK4a mRNA was measured by quantitative real-time PCR and compared to EGFP transduced HUVECs. Beta-actin mRNA expression was used for inter-sample normalisation. MEOX1 is a more potent activator of p16^INK4a than MEOX2. B) A representative western blot showing increased p16^INK4a protein in HUVECs expressing ectopic MEOX1 (MX1), MEOX2 (MX2), but not DNA binding mutant MEOX2^Q235E (Q235E). Total protein was isolated from HUVECs 72 hours after adenoviral transduction at 250 MOI. C) Quantification of the relative amount of p16^INK4a protein compared to EGFP transduced HUVECs. The intensity of the p16^INK4a band was normalized to the tubulin loading control. D) Schematic diagram of the human p16^INK4a promoter luciferase construct used in this paper. The base pair positions are indicated relative to the translational start site. E) Activation of the luciferase reporter gene from the 564 bp p16^INK4a promoter by wild type MEOX1 and MEOX2 but not by the DNA binding domain mutant versions of MEOX2 (MEOX2^Q235E) or MEOX1 (MEOX1^Q220E). Luciferase assays were performed in HUVECs. * Indicates a statistically significant change (p<0.05) when compared to the empty vector or EGFP control. ♦ Indicates a statistically significant difference (p<0.05) between MEOX1 and MEOX2.