Titles and legends to supplemental data
Figure S1 Chemical structures of avicin D and avicin G. Molecular weights as well as differences in the two avicin molecules is shown in the figure.
Figure S2 Early onset of autophagy after avicin D treatment. U2OS/GFP-LC3 cells were pretreated with 50 M zVAD-fmk for 2 h, followed by 2 g/ml avicin D from 0-8 h. The GFP fluorescence was captured with a NIKON ECLIPSE TE2000-E fluorescence microscope following avicin D exposure. The punctate GFP-LC3 fluorescence (autolysosomes/autophagosomes) was induced as early as 2 h after avicin D treatment.
Figure S3 Electron micrograph of U2OS/GFP-LC3 cells treated with avicin D. Proliferating U2OS cells were pretreated with 50 M zVAD for 2 h followed by 2 g/ml avicin D from 0-72 h. The cells were prepared for electron microscopy as described in methods. A large number of autolysosomes/autophagosomes were observed, but the nuclei were unaffected (left two panels, 2,500X and 5,000X). Representative autophagosomes that had double lay membranes are also shown (right two panels, 25,000X and 50,000X). Blue arrows indicate the autolysosomes/autophagosomes. Red arrows indicate the enlarged autolysosomes/autophagosomes and their membranes. U2OS cells grown without serum for 24 h served as positive control.

Figure S4 Avicin D induces autophagy in multiple cell lines. MDA-MB-231, T47D (human breast cancer cell lines), A549 (human alveolar epithelial cancer cell line), SKOV3 (ovarian cancer cell line), and PC3 (prostate cancer cell line) were transfected with GFP-LC3. Twenty-four hours after transfection, cells were exposed to 50 M zVAD-fmk and 2 g/ml avicin D. The GFP fluorescence was captured with a NIKON ECLIPSE TE2000-E fluorescence microscope 24 h following avicin D exposure.

Figure S5 Avicin D induces non-apoptotic cell death in multiple cell lines. MDA-MB-231 (231), T47D, A549, SKOV3, and PC3 were transfected with GFP-LC3 and treated with 50 M zVAD-fmk and 2 g/ml avicin D as described in Figure S4. Seventy-two hours after exposure to zVAD-fmk and avicin D, cells were collected for cell death determination by using trypan blue exclusion assay (a) and for cell viability measurement by using CellTiter-Glo luminescent cell viability assay (b). Data are shown as mean  S.D. (n=3).

Figure S6 Increased ATP generation by methylpyruvate rescues avicin D-induced autophagic cell death.U2OS/GFP-LC3 cells were pretreated with 1 mM methylpyruvate for 8 h, followed by 2 g/ml avicin D for 16 h. (a) ATP levels of cells grown in the presence or absence of methylpyruvate and/or avicin D. Data represent average of three independent experiments ± SD. (b, c) Cell death (b) and cell viability (c) in cells treated with methylpyruvate and/or avicin D for 72 h. Data represent average of three independent experiments ± SD. (d) Autolysosomes/autophagosomes in methylpyruvate- and/or avicin D- treated U2OS/GFP-LC3 cells. The GFP fluorescence was captured with a NIKON ECLIPSE TE2000-E fluorescence microscope 16 h following avicin D exposure.

Figure S7 Time-dependent effect of avicin D on the phosphorylation of AMPK at threonine 172 (T172). U2OS/GFP-LC3 cells were treated with 50 M zVAD-fmk and 2 g/ml avicin D from 0-24 h. After the treatment, cells were collected and western blotting was performed. Fifty micrograms of total protein were loaded each sample. -Tubulin was detected as loading control.

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