Inhibition of SCF^Skp2 induces autophagic cell death. (A) HeLa cells were treated with vehicle, CpdA at 5μM, or CpdA and 200 nM bafilomycin A1 for 4 hours. They were then stained with MDC and visualized by fluorescence microscopy (Nikon FXA, Nikon, Garden City, NY) using Fluor with iris 40×/1.30 lenses. All images were acquired using QImaging Micropublisher CCD camera (Surrey, BC) and were processed using Adobe Photoshop v8 software (Adobe Systems, San Jose, CA). Note the large amount of punctuate MDC staining after treatment with CpdA (middle panel), which is inhibited by bafilomycin (right panel). (B) RPMI 8226 cells were treated with vehicle, CpdA at 10 or 15 μM, or bortezomib at 10 nM, either alone or with 5 μM bafilomycin, as indicated. They were then evaluated for annexin V staining and caspase 3 activation by flow cytometry. Viable cells are annexin V⁻/caspase 3⁻, while those undergoing type I programmed cell death are annexin V⁺/caspase 3⁺, and those undergoing autophagy are annexin V⁺/caspase 3⁻. Data are represented as the mean percentages of each population plus or minus SD from triplicate experiments. (C) Myeloma cells treated under conditions indicated in Figure 4C for 24 hours were analyzed for their content of LC3-I and LC3-II by Western blotting, with β-actin serving as a loading control. (D) RPMI 8226 cells treated with vehicle, bortezomib, or CpdA were then analyzed by transmission electron microscopy as described in “Electron microscopy”. m indicates mitochondria; n, nucleus; and Ap, apoptosomes; white arrows point to autophagic vesicles (Au). The black bar represents 1 μm, and the white bar represents 200 nm.