Fig. 2
3-BrPA activates regulated necrosis and impairs autophagy in bladder cancer cells. (a-b) MTT cytotoxicity assays of T24 (a) and T24-X (b) cells, grown at low confluency (to maximize cells’ sensitization to 3-BrPA; Fig. 1d) and exposed to the indicated doses of 3-BrPA for 24 h, in the absence or presence (pre-incubation for 2 h) of 10 μM PJ-34 (stock in ddH2O). (c-h) MTT cytotoxicity assays of T24 cells, seeded at low confluency and treated with the indicated doses of 3-BrPA for 24 h, in the absence or presence (pre-incubation for the denoted time) of 10 or 100 μM Nec-1 (c), 50 or 100 μM Nec-5 (d), 150 or 300 μM Nec-7 (e), 5 or 10 μM NSA (f), 75 or 100 μM Mdivi-1 (g) and 1 or 10 μM CsA (h). Each inhibitor remained in the growth medium with half of its initial respective concentration for 24 h more, post-pre-incubation. Stock solutions of all inhibitors (c-h) were prepared in DMSO. (c) Two different providers of Nec-1 were independently examined; Sigma-Aldrich (Missouri, USA) and Santa Cruz Biotechnology Inc. (Texas, USA). MTT viability rates obtained from the cocktail of 3-BrPA with each death inhibitor (PJ-34, Nec-1, Nec-5, Nec-7, NSA, Mdivi-1 and CsA) were compared to the respective ones of 3-BrPA alone and, after their normalization with values derived from inhibitor only, versus its cognate solvent, they were presented in fold (x) of inhibitor-induced cell survival increase. (a-h) Data are reported as mean ± standard deviation of triplicates of three independent experiments. *P < 0.001. (e) Light micrographs: T24 cells respectively treated with 150 μM of Nec-7, 100 μM of 3-BrPA, or both, for 24 h. Scale bars: 4 μm. (i) 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 for 24 h. The proteins examined were Atg5, Atg7, Atg12, Beclin-1 and LC3B, while Actin was used as molecule of reference (also, see Additional file 2: Figure S2). (j-k) Quantitative assessment of ADP and ATP cellular contents (ADP/ATP ratio) (j), and lactate cellular levels (k) of T24 cells, seeded at ~60 % confluency and treated with the indicated doses of 3-BrPA for 2 (j) and 4 h (k), respectively (also, see Additional file 2: Figure S2). x: fold of ADP/ATP ratio rise (j) and lactate concentration reduction (k). (j-k) Results are denoted as mean ± standard deviation of triplicates of three independent experiments. *P < 0.001