ROS Production Is Essential for the Apoptotic Function of E2F1 in Pheochromocytoma and Neuroblastoma Cell Lines
Figure 4
E2F1 induces Bax activation, dimerization and translocation to the mitochondria.
(A) PC12 ER-E2F1 cells were transiently transfected with an YFP-Bax plasmid, serum-deprived and treated with (OHT) or without (control) OHT for 3 hours. YFP-Bax localization was achieved by immunofluorescence as indicated in Material and Methods. Red signal represents the mitochondrial staining with MitoTracker and green signal represents Bax-YFP localization. (B) Quantification of the punctuated mitochondrial clusters of YFP-Bax from the experiments described in A. (C) PC12 ER-E2F1 cells were serum-deprived and treated with (OHT) or without (control) OHT for 3 hours and active Bax was detected by immunofluorescence using anti-Bax 6A7 clone antibody. Red signal represents the mitochondrial staining with MitoTracker and green signal represents active Bax. (D) Quantification of the positive anti-BAx 6A7 from experiments described in C. (E) PC12 ER-E2F1 cells were treated with OHT at the indicated times and harvested and the mitochondria fraction was isolated. Crosslinking reactions were performed on the mitochondrial pellets as indicated in Materials and Methods. Samples were subjected to Western blot analysis and probed with anti-Bax antibody. (F) PC12 ER-E2F1 cells were treated with 50 µM of Bax-inhibiting peptide in the presence or in the absence of OHT. At 72 hours, apoptotic cells were identified by TUNEL and counted against 100 nuclei identified by Hoescht staining in randomly taken photographs. Results are presented as Mean ± SEM, for n = 3. Statistically significant differences were obtained comparing with cells untreated with Bax-inhibiting peptide. Student’s t-test value of **p<0,01 was considered statistically significant.