Fig. 10

Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were transfected with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0 < C1 ≤ 1 μm²; medium: 1 < C2 ≤ 3 μm²; large: C3 > 3 μm²) are reported. Cells were analyzed using a fluorescence microscope at 100× magnification and ImageJ JavaScript software, as described by Fassina et al. [50]