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Fig. 3 | Molecular Cancer

Fig. 3

From: 3-BrPA eliminates human bladder cancer cells with highly oncogenic signatures via engagement of specific death programs and perturbation of multiple signaling and metabolic determinants

Fig. 3

p53 is not engaged in genotoxic responses of bladder cancer cells to 3-BrPA: 3-BrPA and PRIMA-1 cooperative actions. (a-b) Representative (three independent experiments) Western blotting profiles of whole-cell protein extracts derived from RT4, T24 and T24-X cells, grown at ~60 % confluency and exposed to the indicated doses of 3-BrPA (a) or Doxorubicin (b) for 24 h. The proteins examined were p-H2A.X-Ser139, H2A.X, p-p53-Ser15, p-p53-Ser392 and p53, while Actin was used as molecule of reference (also, see Additional file 3: Figure S3). p: phosphorylation. (c) Representative (three independent experiments) Western blotting profiles of whole-cell protein extracts obtained from untransfected (−) and transiently transfected (CMV-p53 or CMV control vector) T24 cells, treated (48 h post-transfection) with the indicated doses of 3-BrPA (left panel) or Doxorubicin (right panel) for 24 h. The proteins examined were p-p53-Ser15 and p53, while Actin was used as molecule of reference. (d) MTT cytotoxicity assays of T24 cells, after their transient transfection with either CMV-p53 expression vector (gray bars) or CMV control (empty) vector (white bars) and subsequent exposure (48 h post-transfection) to the indicated doses of 3-BrPA for 24 h. (d) Data are reported as mean ± standard deviation of triplicates of three independent experiments. *P < 0.001. (e) MTT cytotoxicity assays of RT4, T24 and T24-X cells, grown at ~60 % confluency and treated with the indicated doses of PRIMA-1 (stock in ddH2O) for 6 h and half of its initial respective concentrations for 24 h more. (f) MTT cytotoxicity assays of RT4, T24 and T24-X cells, seeded at ~60 % confluency and exposed to the indicated doses of 3-BrPA in the presence of PRIMA-1 (pre-incubation for 6 h, with 100 or 200 μM) for 24 h (5, 6, 8 and 9). PRIMA-1 remained in the growth medium, containing (5, 6, 8 and 9) or not (4 and 7) 3-BrPA, with half of its initial respective concentrations for 24 h more, post-pre-incubation (also, see Fig. 3e). 3-BrPA (50 or 75 μM) alone was added to the cells for 24 h (2 and 3; also, see Fig. 1). PI: pre-incubation. (e-f) Results are denoted as mean ± standard deviation of triplicates of three independent experiments. *P < 0.001

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