Effect of treatments on MIF secretion, intracellular content and gene expression. A. MIF secretion – Conditioned medium following 24 h growth was assayed for MIF content by ELISA. Data are expressed as culture medium MIF concentration in pg per 105 cells. All treatments decreased MIF in the culture media. B. Western blot of anti-MIF treated conditioned medium following 24 h treatment- 12 kDa MIF band is present in all samples. Lane 1, DMEM 1% BSA; Lane 2, non-specific mouse IgG1; Lane 3, anti-MIF antibody treatment. C. Intracellular MIF content – Changes in intracellular MIF 24 h post treatment. Data are expressed as cleared cell lysate MIF concentration in pg per 105 cells. Note that HA treatment increased, while MIF antisense treatment decrease the concentration of MIF in the cell lysates. Anti-MIF treatment did not produce a significant effect. D. MIF gene expression -MIF mRNA content was quantified in cells following 24 h and the indicated treatment. Data are expressed as a relative intensity ratio. PCR band intensity was determined from the formula, total intensity = area × average intensity. The relative intensity ratio is determined from the total intensity of gene specific PCR product band divided by the 18 S rRNA band intensity (internal standard). All treatments resulted in a decrease in MIF mRNA with the greatest effects seen following MIF anti-sense treatment. (* – p < 0.05, ** – p < 0.01, *** – p < 0.001).