Fig 2: Role of MDM2 in LA-triggered p53-depletion in HCT116 cells. (A) Time course of MDM2 gene expression. Cells were exposed to LA as indicated. EtOH was used as solvent control (0 µM LA), while 5-FU was used as positive control. (B,C) HCT116 were treated with increasing doses of LA for 24 h (B) or 48 h (C) either with or without the MDM2 inhibitor Nutlin-3a. In addition, 5-FU (5 µM) was used as positive control for p53 and p53-dependent MDM2 and p21 induction. Cells were analyzed by SDS-PAGE followed by western blot detection of MDM2, p53 and p21. Either Hsp90 or ß-Actin served as loading control. (D–F) Densitometric quantification of MDM2 (D), p53 (E), and p21 (F) normalized to the respective loading controls obtained from three independent experiments as described and shown in (B). Data are displayed as mean + SEM of three independent experiments.

Fig 3: p53ß/? isoforms are resistant to MDM2-mediated degradation.A, Western blot analysis showing p53a and p53ß/? expression in A549 cells treated with indicated drugs. B and C, quantification of p53a and p53ß/? protein expression, respectively, in A549 cells treated with the indicated drugs. D, Western blot analysis of A549 cells treated with indicated siRNAs and either dimethyl sulfoxide or nonsense-mediated decay inhibitor (NMDi). E–G, quantification of MDM2, p53a, and p53ß/? protein expression, respectively, in A549 cells treated with the indicated siRNAs and either dimethyl sulfoxide or NMDi. Western blot analysis quantifications shown (n = 3) are from three independent experiments. Mean ± SD, p values, two-tailed t tests, * =0.05, ** <0.01, *** <0.001, **** <0.0001, NS, not significant. Western blots shown are a representative of three independent experiments. NMDi, nonsense-mediated decay inhibitor.

Fig 4: Morphologic characteristics and antigenic marker expression of the novel DDCS2 cell line. (A) DDCS2 cells display spindle to polygonal shape in vitro under light microscopy (200×, scale bar: 50 µm). (B) The growth curves of DDCS2 were plotted using cells plated in 96-well plates. (C) Western blots revealed the expression of vimentin, cytokeratin, p53, and MDM2 in various sarcoma cell lines compared with two epithelial carcinoma cell lines. Relative expressions of vimentin, cytokeratin, p53, and MDM2 were figured in different sarcoma and epithelial carcinoma cell lines compared with ß-actin. (D) Immunofluorescence analysis of vimentin was conducted in DDCS2, CS-1, and SW1353 cell lines (200×, scale bar: 50 µm).

Fig 5: EPO increases Mdm2 expression after DNR or nutlin-3a treatment and suppresses nutlin-3a-induced apoptosis.Western blot showing p53 and Mdm2 protein levels in cells treated with DNR (a, b) or with nutlin-3a (c, d) in the presence or absence of EPO. In (a) and (b), DA3/EPOR cells were cultured in the absence of EPO for 1 h followed by 10 h of treatment with DNR (0.25 µM) with or without EPO (1 U/ml). In (c) and (d), cells were cultured in the absence of EPO for 0.5 h followed by 24 h of treatment with nutlin-3a (10 µM) with or without EPO (1 U/ml). Protein expression levels were determined by western blot analysis using the indicated antibodies. The HDM2-323 antibody recognizes full-length MDM2 and the SMP14 antibody recognizes the MDM2 p60 cleavage product. (e) Cells were treated as in (c, d) and the proportion of cells undergoing apoptosis (cells having <2 N DNA content) was measured by flow cytometry after staining with propidium iodide