Supplementary Figure legends. Supplementary Methods

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1 Supplementary Methods Transmission electron microscopy. For transmission electron microscopy (TEM), the cell populations were rinsed with 0.1 Sorensen s buffer (ph 7.5), fixed in 2.5% glutaraldehyde for 1.5 h, and subsequently dehydrated and embedded in Spurr s resin. The block was then sectioned into nm ultra thin sections and picked up on copper grids. For routine analysis, ultrathin sections were stained with 2% uranyl acetate and lead citrate. Electron micrographs were obtained using a transmission electron microscope (Joel JEM-1230 equipped with a Gatan UltraScan 4000SP 4K 4K CCD camera). Plasmids, transfection and RNA interference. Cells were cultured in six-well plates and transfected at 80% confluence with Oligofectamine reagent (Invitrogen), in the presence of 100 nm of sirnas specific for human Beclin-1, atg5, hvps34, atf6 and ire1 with appropriate sirna controls. Transient transfections with plasmids were performed with Lipofectamine 2000 reagent (Invitrogen) and cells were used 48 h after transfection for autophagy and apoptosis studies. Assessment of Cytosolic Ca2+ Levels. DU-145 Cells were plated in 24-well plates and transfected with or without calbindin and Calcium levels were measured from cells loaded with the calcium-sensitive fluorescent dye fura-2am (Molecular Probes, Junction City, OR) as described by the manufacturer. Briefly, after Ad.mda-7 treatment for different times DU-145 cells were loaded with fura-2am (5μM) for 30 min at 37 C and fluorescence was measured using Victor3 (PerkinElmer, Waltham, Massachusetts). To quantify the extracellular calcium, fluorescence was measured using, excitation of 340nm and 380nm and the ratio of fluorescence intensities detected at ~510 nm was determined (Thomas and Delaville, 1991). From this ratio, the level of intracellular calcium can be accurately estimated. Supplementary Figure legends Supplementary Figure S1. Electron microscopic analysis of autophagy. (A G) Selected micrographs showing the assembly and progression of autophagic vesicles. (A) Sequestration of cytoplasm by pre-autophagasome (arrow); (B) Cup-shaped membranous structures (arrow) in the cytoplasm; (C) An autophagosome (arrow), containing organelles undergoing degenerative changes; (D) A close contact of autophagosome with lysosome (arrow); (E) Docking and fusion

2 of autophagosome with lysosome (arrow); (F) Typical autolysosomes containing remnant of digested organelles or cytoplasm (arrow) (G) Features of late autophagic cell showing membrane blebbing (arrow head) and autolysosome (arrow). Scale bars: (A, B and C), 0.5 μm; (D, E), 1 μm; (F,G), 1 μm. Supplementary Figure S2. MDA-7/IL-24 induces autophagy in prostate cancer cells. Prostate cancer (PC-3, LNCaP) and P69 normal immortal prostate epithelial cells were infected with Ad.vec or Ad.mda-7 (100 pfu/cell) for 24 h and LC3 expression was determined by Western blotting. Supplementary Figure S3. (A) DU-145 cells were transfected with the indicated sirnas and protein down-regulation was determined by immunoblotting; (B) MDA-7/IL-24 regulates the levels of specific chaperone protein expression. Cells were infected with Ad.vec or Ad.mda-7 (100 pfu/cell) for 24 h and changes in BiP/GRP78, GRP94, GADD153, total eif2α, and p-eif2α proteins were quantified by Western blot analyses. (C) DU-145 cells were infected with 100 pfu/cell of Ad.vec or Ad.mda-7 for different times and ceramide generation was measured as described in Material and methods. Values reported are the mean ± S.D. of three independent experiments. *, p < 0.05 versus control cells. (D) Functional assay of dominant negative PERK. Cells were treated with 1 µm of Thapsigargin (TG) for 24 h and analyzed by Western blotting for total eif2α and p-eif2α as a downstream target of PERK. (E) LC3-II expression 24 h after administration of the indicated sirnas and Ad.mda-7 infection (100 pfu/cell) was determined by immunoblotting. Supplementary Figure S4. (A) DU-145 cells were infected with 100 pfu/cell of Ad.vec or Ad.mda-7 and immunoprecipitated with anti-mda-7/il-24 or anti-beclin-1 followed by immunoblotting with anti-beclin-1, anti-mda-7/il-24, anti-bcl2 or anti-bcl-xl antibodies. (B) DU-145 cells were infected with 100 pfu/cell of Ad.vec or Ad.mda-7 for different times and calcium mobilization was measured as described in Material and methods. Values reported are the mean ± S.D. of three independent experiments. *, p < 0.05 versus control cells.

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6 Supplementary Figure-4