(A) Protein extracted from bafilomycin-treated or untreated NTLV and ZKSCAN3-shRNA cells was subjected to Western blotting using anti-LC3 and Actin antibodies. The graph shows quantification (mean ± s.d.) of LC3-II band intensity relative to Actin. *P<0.05 (B) Representative live cell images of cells stably expressing a GFP-RFP-LC3 fusion protein. (C) NTLV and ZKSCAN3-shRNA cells were transiently transfected with a plasmid encoding GFP-Neo fusion protein. After 48 h, ZKSCAN3-shRNA cells expressing GFP-Neo were treated or untreated with bafilomycin (5 nM, lane 3) for 24h. Subsequently after total 72 h, protein was extracted and Western blotted using anti-GFP and anti Actin antibodies (D) UC13 and RKO cells were either transiently transfected with an empty vector (pcDNA3.1) or a streptavidin-flag-tagged-ZKSCAN3 expressing vector. After 72h, cells were treated, where indicated, with rapamycin (6h/200 nM), extracted and immunoblotted for LC3 and Actin.