<p>(A–D) tfLC3-MCF7 cells (A, B) or MCF7 cells (C, D) were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA (CT) or indicated siRNA pools (3×6.67 nM). (A, B) After 48 h, tfLC3-MCF7 cells were analyzed by confocal microscopy. Representative images (A; <i>Bars</i>, 10 µm) and quantification of puncta (B) are shown. Raptor siRNA (RPTOR) served as a control for increased autophagic flux. Closed arrows indicate AVd, open arrows indicate AVi. (C) After 60 h, the level of p62/SQSTM1 (p62), which is degraded by autophagy, was examined by Western blot. Rapamycin (20 nM, 4 h) was used to induce autophagy. Numbers represent p62 levels as percentage of the level in untreated control siRNA-transfected cells. (D) <i>Top</i>, After 60 h, MCF7 cells were treated with 100 µg/ml Alexa Fluor 488-dextran (dextran-488) for 1 h and analyzed by flow cytometry (FL1-H). The threshold for high intensity staining was defined so that 88% of control siRNA-transfected cells were below. <i>Bottom</i>, Example histograms showing dextran-488 content of cells transfected with control or KIF20A siRNA. M1 = gate for high intensity staining. Values represent means + SEM of 20 cells in one representative experiment (B, n = 3) or means + SD of three independent experiments (D). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected cells.</p
