Transfer of ATG7 siRNA blocked tBHP-induced cell death in Bβ2#5 clone. A total of 1 × 106 exponentially growing cells of both Bβ2#5 and Bβ1#1 clones were transfected with either scrambled siRNA (SO) control or ATG7 siRNA, treated with tBHP and analyzed. (A) Knocked-down ATG5 expression by the increased ATG7 siRNA Cell lysates of Bβ2#5 clones were probed with ATG7 antibody and reprobed with β-actin antibody to confirm equal loading of the proteins. The blots were then detected with ECL system. (B) Cell growth Both Bβ2#5 and Bβ1#1 clones were treated with tBHP for 48 h following transfection with either SO control or ATG7 siRNA. The viable cells were determined by trypan-blue staining. (C) Determination of apoptotic cells Distribution of annexin V binding in both clones was determined by flow cytometry after 48 h treatment with 50 nM of tBHP. The results represented averages of three independent experiments and error bars standard errors. Data are represented as the mean ± standard errors of three independent experiments, each performed in duplicate. *P < 0.05, significance of difference as compared with the control group. (D) Western blot analysis with LC3 antibody Development of autophagy by tBHP was analyzed by western blot analysis by incubating the blot with LC3 antibody with β-actin antibody as loading control. The elevated LC3-II as visualized the 16-kDa form of LC3 specific for membranes of the formed autophagosomes by tBHP in Bβ2 #5 clones was suppressed by the increased concentrations of ATG7 siRNA.
Cheng et al. BMC Cell Biology 2009 10:91 doi:10.1186/1471-2121-10-91