Figure 1.
The effect of Serdemetan on p53 levels.
A) Chemical structure of Serdemetan (SE) and Nutlin3. Western blot of U87 cell lysates treated with increasing concentrations of Nutlin3 or Serdemetan for 6 h to detect p53, Mdm2 and GAPDH. B) Western blot of p53, p21 and GAPDH from U373, SF767, and U87. Whole cell lysates were prepared from cells treated with either DMSO or 10 µM Serdemetan (SE) for 6 h.
Figure 2.
Detection of HIF1α in response to Serdemetan treatment.
A) Western blot of HIF1α, Mdm2 and GAPDH from SF767, U87 and U373 cellular extracts. Cells were subjected to treatment of either 21% oxygen or hypoxic (1%) conditions for 6 h with 10 µM Serdemetan (Se) or DMSO (C). B) Western blot analysis of HIF1α, tubulin, and PARP from cytoplasmic (C) and nuclear (N) extracts of SF767 cells treated with 10 µM of Serdemetan (Serd) or DMSO (Control). C) Western blotting was performed for HIF1α, tubulin, and PARP as described above with the addition of pretreatment with 10 µM MG132 for 16 h.
Figure 3.
Analysis of HIF1α targets in response to Serdemetan.
A) Luciferase assay of U87 cells transfected with the 4X HRE luciferase construct (left panel) or real time PCR for vegf from U87 cells (right panel) treated with 10 µM Serdemetan or DMSO under hypoxic conditions. B) Western blot of VEGF levels in SF767 and U373 cells. Cells were treated with 10 µM Serdemetan or DMSO control under hypoxic conditions. C) Western blot analysis of enolase, Glut1, PGK1/2 and GAPDH from U87 and U373 cells. Cells were treated with 30 µM of Serdemetan and subjected to hypoxia for 24 h.
Figure 4.
Survival of cells treated with Serdemetan.
A) Colony forming assay was performed on SF767, U373, and U87 cells under hypoxia for 48 h with DMSO or Serdemetan. B) Quantitation of the colony-forming assay by measuring the absorbance at 595 nm as percent change from DMSO. Error bars represent standard deviation as calculated from the mean (n = 3).