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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Mol Cancer Ther. 2015 Oct 22;14(12):2850–2863. doi: 10.1158/1535-7163.MCT-15-0237

Figure 3. Effects of single agent and combination treatment on Mdm2 and dependency of treatment-mediated cell death on p73α.

Figure 3

(A) TMD231 cells were transiently transfected with siControl or sip73 siRNA and treated with Vehicle (Veh), 15 µM Nutlin-3a (Nut), 15 µM carboplatin (Carb), or 1:1 combination (Combo) for 24 hours and levels of Mdm2, MdmX, p73α, p53, p21, PUMA, and GAPDH determined by Western blot. Densitometry for each protein was quantified by ImageJ and shown below each protein relative to siControl Vehicle-treated cells. (B) TMD231 cells transfected with sip73 or siControl (siCon) were treated with 15 µM Nutlin-3a (Nut), 15 µM carboplatin (Carb) or Nutlin-3a/carboplatin (Combo) for 3 days and IC50 values for inhibiting cell growth were determined after methylene blue staining (*p<0.05, vs siControl, Student’s t-test, n=5 independent experiments). (C–D) Cells were not treated (Untx) or treated with Vehicle (Veh), 15 µM Nutlin-3a (Nut), 15 µM carboplatin (Carb), or 1:1 combination (Combo) for 6 hours. Mdm2 levels of (C) whole cell lysates (WCL) and the (D) chromatin fraction (Chromatin) were determined by Western blot. β-actin (WCL) and H2AX (chromatin) served as loading controls. Densitometry is shown above the blots. Data are representative of 2 independent experiments. (E) γH2AX foci formation treated with Vehicle (Veh), 7.5 µM Nutlin-3a (Nut), 7.5 µM carboplatin (Carb), or 1:1 combination (Combo) (**p<0.01 vs Vehicle, ***p<0.001 vs Vehicle, ††p<0.01 vs Nut, †††p<0.001 vs Nut, #p<0.05 vs Carb, ###p<0.001 vs Carb at same time point, Holm-Sidak post hoc test, n=5 fields per group). (F) γH2AX levels normalized to β-Tubulin following Vehicle (Veh), 15 µM Nutlin-3a (Nut), 15 µM carboplatin (Carb), or 1:1 combination (Combo) in TMD231 cells (**p<0.01 vs Vehicle, ***p<0.001 vs Vehicle, ## p<0.015 vs Nut, ###p<0.001 vs Nut, and @p<0.01 vs Carb at the same time point, Holm-Sidak post hoc test, n=3 independent repeats). (G) Mdm2 levels normalized to β-Tubulin following Vehicle (Veh), 15 µM Nutlin-3a (Nut), 15 µM carboplatin (Carb), or 1:1 combination (Combo) in TMD231 cells (**p<0.01 vs Vehicle, ***p<0.001 vs Vehicle, @@@p<0.001 vs Carb, and ††p<0.01 vs Nut at the same time point, Holm-Sidak post hoc test, n=3 independent repeats).