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1 Box S1: Methods for studying lysosomal function and integrity Volume and distribution of the acidic compartment. Acridine orange is a metachromatic fluorochrome and a weak base that accumulates in the acidic compartment of the cell mainly composed of lysosomes. It exhibits red fluorescence when highly concentrated in acidic vesicles and green fluorescence when less concentrated in other parts of the cell. The total volume of the acidic compartment can hence be measured as the red fluorescence by flow cytometry or confocal microscopy 1,2. The decrease in the red and the increase in the green fluorescence intensity reflect the loss of the lysosomal ph gradient. A series of commercial fluorophores, LysoTracker and LysoSensor probes ( are also well suited for staining of the acidic compartment. More specific visualization of the lysosomal compartment can be achieved by staining with antibodies against lysosomal membrane proteins 3. A B EM images of murine embryonic fibroblasts loaded with gold-coupled albumin before the 5 hour treatment with 4 M cycloheximde alone (A; note gold particles inside the vesicle) or with 10 ng/ml TNF (B; note gold particles freely in the cytoplasm). Scale bars are 100 nm. (Courtesy of Mads Gyrd-Hansen and Lone Bastholm) Lysosomal membrane permeabilization (LMP). Lysosomes that have lost part of their contents appear ultrastructurally normal 4. However, LMP can be visualized by immunostaining of lysosomal hydrolases 5,6, or microscopic analysis of cells fed with fluorescence-labelled dextrans or transfected with GFP-labelled lysosomal lumen proteins 7,8. Background staining limits the specificity of these methods, whereas electron microscopy of cells loaded with gold-coupled albumin is practically devoid of background allowing the detection of cells with only few permeabilized lysosomes (Fig. A and B). LMP can be quantified by measuring the percentage of the total acid hydrolase

2 (e.g. cysteine cathepsin) activity found in the cytosol of cells with intact plasma membranes 9. Secretion of lysosomal contents to the extracellular space Lysosomal exocytosis can be detected by immunoprecipitation or activity measurements of lysosomal hydrolyses from the culture supernatant or alternatively by surface staining for lysosomal membrane proteins that are transiently localized to the plasma membrane upon exocytosis 10.

3 Box S2: Methods for detecting autophagy A B EM images of MCF-7 breast cancer cells treated with 100 nm EB1089 vitamin D analogue for 3 (A) or 4 (B) days. Scale bars are 200 nm. (Reproduced with permission from REF. 11 ) Autophagic vacuoles (AV) can be detected by electron microscopy (EM) as double membrane-surrounded vesicles containing cytoplasmic material and organelles (Fig. A). In addition, such vesicles can be detected by transfection with an LC3-green fluorescent protein (GFP) fusion construct. LC3-GFP is normally diffusively distributed through the entire cell, yet concentrates in autophagic vacuoles upon autophagy induction, a phenomenon that can be detected in living cells (by videomicroscopy) or in fixed cells cultured in vitro or recovered from mice expressing an LC3-GFP transgene 15,16. Limited proteolysis of endogenous LC3 to the LC3II isoform occurs during AV formation and can be detected by immunoblot analysis 15. The fusion of autophagosomes and lysosomes can be detected by EM (Fig. B), two-colour staining for molecular markers of lysosomes (LAMP1, LAMP2, LIMP1, cathepsins) and autophagosomes (LC3-GFP) or their content (immunodetection of endogenous mitochondrial and/or ER proteins or transfection of fluorescent organelle markers such as mitochondirialdsred) 14,17,18. Overlapping signals determined by confocal fluorescence microscopy can be interpreted as autophagolysosomes. Note that all these morphological analyses have to be completed by biochemical determination of the increased turnover of long-lived proteins. This type of analysis is typically performed by pulse-chasing cells with radioactive aminoacids and determination of their half live in the absence or presence of autophagy inhibitors. It is also important to note that lysotracker probes and the flurochrome monodansylcadaverine are not specific for autophagosomes, but rather stain the entire acidic compartment 11.

4 References 1. Olsson, G.M., Rungby, J., Rundquist, I. & Brunk, U.T. Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry. Effect of silver lactate and hypotonic conditions. Virchows Arch B Cell Pathol Incl Mol Pathol 56, (1989). 2. Nylandsted, J. et al. Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization. J Exp Med 200, (2004). 3. Eskelinen, E.L., Tanaka, Y. & Saftig, P. At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol 13, (2003). 4. Brunk, U.T. & Ericsson, J.L. Cytochemical evidence for the leakage of acid phosphatase through ultrastructurally intact lysosomal membranes. Histochem J 4, (1972). 5. Boya, P. et al. Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion. J. Exp. Med. 197, (2003). 6. Roberg, K., Johansson, U. & Ollinger, K. Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress. Free Radic Biol Med 27, (1999). 7. Bidere, N. et al. Cathepsin D triggers Bax activation, resulting in selective AIF relocation in T lymphocytes entering the early commitment phase to apoptosis. J Biol Chem 278, (2003). 8. Roberts, L.R. et al. Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes. Gastroenterology 113, (1997). 9. Foghsgaard, L. et al. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J. Cell Biol. 153, (2001). 10. Reddy, A., Caler, E.V. & Andrews, N.W. Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell 106, (2001).

5 11. Høyer-Hansen, M., Bastholm, L., Mathiasen, I.S., Elling, F. & Jäättelä, M. Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death. Cell Death Differ (2005). 12. Levine, B. & Klionsky, D.J. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 6, (2004). 13. Shintani, T. & Klionsky, D.J. Autophagy in health and disease: a double-edged sword. Science 306, (2004). 14. Kirkegaard, K., Taylor, M.P. & Jackson, W.T. Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol 2, (2004). 15. Kabeya, Y. et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, (2000). 16. Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T. & Ohsumi, Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell. 15, (2004). 17. Boya, P. et al. Inhibition of macroautophagy triggers apoptosis. Mol Biol Cell in press(2005). 18. Gonzalez-Polo, R.-A. et al. The apoptosis/autophagy paradox. Accumulation of autophagic vacuoles triggers apoptosis. in press(2005)..