ASK induces chromatin fragmentation and phosphatidylserine translocation in BCa cell lines. (A) UMUC-3, RT4 and T24 cells were transfected with Andes-1537 or ASO-C or left untreated (NT) for 48 h and subjected to TUNEL assay. Andes-1537 transfection induced an increase in TUNEL-positive cells. Bars = 50 µm. (B) Quantification of TUNEL positive cells in in all 3 lines. (C) BCa cell lines were analyzed by flow cytometry, revealing a higher population of Annexin V-positive cells in samples treated with Andes-1537. (D) Quantification of Annexin V-positive cells in the three cell lines, as compared to controls (NT and ASO-C). (E) Total lysates of UMUC-3 cells treated as in (A) were subjected to Western blot, revealing triggering of apoptosis through cleavage of procaspase-3 and drastic downregulation of Bcl-xL and survivin by ASK, compared to controls (NT and ASO-C). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Andes-1537 vs. ASO-C.