Figure 5

MIF modulates the PI3K/Akt signalling pathway in melanoma cell lines. MIF expression was depleted in a panel of six melanoma cell lines as described before. Three days after transfection, the cellular levels of MIF, Akt and key cell cycle regulators were measured using Western blotting. Densitometric signals were calculated for each protein band using the MultiGauge software package (Fuji Life Sciences) and used to determine relative expression following depletion of MIF. (A) Representative Western blots showing specific immunoreactive bands for MIF in the indicated melanoma cell lines. The normalised ratio of expression of MIF was determined by dividing MIF levels after depletion against control levels. (B) Levels of phosphorylated Akt (Ser 473) and total Akt where the ratio represents the relative phospho-Akt levels compared to total Akt. (C) Levels of the cell cycle regulators cyclin D1, CDK4 and the cyclin-dependent kinase inhibitor p27. Each ratio was determined by dividing the optical density of the specific band by the GAPDH value. The results shown were consistent across at least 3 independent experiments. (D) Dual parameter plot comparing the degree of inhibition of proliferation after MIF knockdown (effects on the proportion of cells entering S-phase; Figure 3) with the corresponding effects on the level of Akt activity observed (relative levels of pAkt; Figure 4A).