Figure 2.

RACK1 depletion-induced autophagy is non-canonical. (a) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies (left panel). Intensities of Atg5/12 and Beclin1 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells were plotted (right panel). (b) HT1080 cells were transfected with siRNAs against control or RACK1 in combination with or without Beclin1 siRNA (100 pmol). After 48 h, these cells were analyzed by immunoblotting using the indicated antibodies. (c) HT1080 cells were transfected with control or Atg5 siRNA (100 pmol). After 24 h, the cells were re-transfected with control or RACK1 siRNA. After a further 48 h incubation, the cell extracts were subjected to immunoblot analysis using the indicated antibodies. (d) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies. (e) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 24 h. Extracts of siRNA-transfected cells were pretreated with rapamycin 1 μM for 24 h, followed by immunoblot analysis using the indicated antibodies. Intensities of LAMP1 and LAMP2 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells were plotted. *P<0.05, **P<0.01 (Student’s t-test)