Fig. 4.

MPT0B291 induces senescence and diminished the expression stemness-related markers in both GBM spheroids and TMZ-resistant (TMZ-R) cells. (A) Mitotic P3 cells were released into the cell cycle by removing nocodazole from the culture, and then harvested at different time points as indicated. Cells were then fixed for cell cycle progression assay using flow cytometry. The percentages of cells in G1 phase and G2/M phase are shown in right panel. (B) U87MG spheroids (S/S) were treated with DMSO or MP. At 4 days posttreatment, senescence was examined using SA β-gal staining. Photomicrographs of spheroids were randomly selected in microscopic fields, and SA-β-gal positive cells were counted. (C) Cells dissociated from U87MG spheroids via trypsinization were grown in soft agar, followed by treatment with different doses of MP every 4 days. After 3 weeks of incubation at 37°C, the colonies that arose from these single cells were photographed randomly, and a histogram of average colony numbers was plotted after performing the experiment in triplicate. (D and E) Cells, as indicated, after 2 days of MP treatment were harvested, and the cell lysates were analyzed using IB with the indicated antibodies. (t-test: **P < 0.01)