Figure 5.

17AAG-induced apoptosis and growth defects are significantly rescued by excess MIF. (A and C) U2OS (A) and SW480 (C) cells were transiently transfected with increasing amounts (indicated as wedges) of MIF expression plasmid (ect MIF) or 0.8 µg empty control vector per well in 12-well plates. At day 1 after transfection, cells were treated with 5 µM 17AAG for 24 h, or left untreated, and stained with Annexin and 7-AAD to count cells in early and late apoptotic cell phases by flow cytometry. Error bars indicate the mean of three independent experiments. (B and D) U2OS (B) and SW480 (D) cells were transiently transfected with MIF expression plasmids as in A and C. At day 1 after transfection, 5 × 10⁴ cells per 12-well plate were seeded (d0) and cultured for another 24 h. Cells were then treated with 5 µM 17AAG for 24 h (time interval indicated by vertical dashed lines) or left untreated. During subsequent culturing, cell numbers (U2OS) or cell confluence (SW480) was measured by CELIGO Cytometer using 49 squares per well. Error bars indicate the mean of two independent experiments in duplicates each. Time is in days (d).