Figure 2.

TM increases autophagic sequestration and degradation activity in LNCaP cells. A, LNCaP cells were treated for 24 h, as indicated, with Baf and Torin1 included the last 3 h only, followed by determination of LDH sequestration (mean ± S. E. (error bars), n = 4). Bottom, the LDH sequestration assay measures autophagic sequestration of cytosol (autophagosome formation), using endogenous LDH as a cargo probe and a crude fractionation protocol to separate cytosolic (nonsedimentable) from sedimentable LDH (38–40, 46). In the presence of the lysosomal inhibitor Baf, the degradation of the LDH that resides inside autophagosomes is blocked, allowing specific analysis of autophagic sequestration activity. B, LNCaP cells transfected with siCtrl or siULK1 + siULK2 were treated for 24 h, as indicated, with Baf included the last 6 h only. LLPD was measured at 18–24 h (mean ± S.E., n = 3). Bottom, the LLPD assay measures endogenous degradation of long-lived proteins and is a classical method for monitoring autophagic flux (33, 48, 49). The method is a true end point assay of the autophagic pathway (i.e. it monitors the product of protein degradation). The proportion of autophagic–lysosomal LLPD is determined by treatment with Baf as well as by RNAi-mediated silencing of key autophagy-related genes, such as ULKs (49). Red dots represent individual data points. Statistical significance was evaluated using repeated measures one-way ANOVA for A and repeated measures two-way ANOVA for B. *, p < 0.05; **, p < 0.01; ***, p < 0.001. N.S., not significant.