Figure 4.

Basic pH-induced autophagy is mediated by Atg5, Vps34 and Beclin 1. MCF-10A cells were reverse-transfected with indicated siRNAs and cultured for 48 h. (A) Knockdown efficiency was measured by real-time PCR assay. siGL2, which targeted the luciferase gene in the pGL2 construct, was used as the negative control. (B) Cells were incubated in medium of indicated pH in the presence or absence of lysosomal inhibitors Pepstatin and E64d. After 6 h, LC3 level was measured by protein gel blotting followed by densitometry quantification using the Image J program. The calculation of the relative LC3-II intensity was performed as described in Materials and Methods.