Supplemental Material 1

Supplemental Material 1

List of antibodies used

The following primary antibodies were used: a rabbit polyclonal caspase-3 antibody (#9662, Cell Signaling Technology, Danvers, MA, USA), a mouse monoclonal caspase-8 antibody (No 804-242, Alexis, San Diego, CA, USA), a mouse monoclonal XIAP antibody (No 610763, BD Transduction Laboratories, San Jose, CA, USA), a goat polyclonal Bid antibody (AF860, R&D Systems), a mouse monoclonal LC3 antibody (Abgent, San Diego, CA, USA), a rabbit polyclonal LC3 antibody (L8918, Sigma-Aldrich), a rabbit polyclonal p62 antibody (sc-25575, Santa Cruz Biotechnology), a mouse monoclonal RIPK1 antibody (610458, BD Transduction Laboratories), a mouse monoclonal FADD antibody (No. 610399, BD Transduction Laboratories), a rabbit polyclonal PARP antibody (#9542, Cell Signaling Technology, Danvers, MA, USA), a mouse monoclonal Bcl-2 antibody (sc-509, Santa Cruz Biotechnology), a rabbit polyclonal green fluorescent protein antibody (PC408, Calbiochem), a mouse monoclonal α-spectrin antibody (MAB1622, Chemicon, Millipore, Molsheim, France), a mouse monoclonal Atg5 antibody (A2859-200 Sigma-Aldrich), a mouse monoclonal α-tubulin antibody (T9026, Sigma-Aldrich), a mouse monoclonal β-actin antibody (A5441, Sigma-Aldrich) and a mouse monoclonal porin antibody (No 539536, Calbiochem). Anti-mouse IgG, anti-rabbit IgG or anti-goat IgG peroxidase conjugated secondary antibodies (AP124P, AP132P, AP106P, Millipore) were used at a dilution of 1:5000 for 1 h.

List of siRNA sequences used

Procaspase-3 (AAU GAC AUC UCG GUC UGG UAC) (1), procaspase-7 (AAG CAA UGG GUC ACU CAU UAA [dT]) (2), procaspase-8 (AAU UCG GAA GAG CAG CUC C[dT]) (3), FADD (GAG CCA GCC UCC UCC AAU C[dT]) (4), XIAP (AAG UGG UAG UCC UGU UUC AGC) (5), Atg5 (GCA ACU CUG GAU GGG AUU G[dT]) (6), scrambled control siRNA (ACU UAA CCG GCA UAC CGG C[dT]).

Flow cytometry settings

For FRET measurements, CFP was excited with the 405 nm laser and fluorescence emission was detected through 455 nm (Partec Cyflow) or 450/50 (BD LSR II) band pass filters. YFP was excited with the 488 nm laser and fluorescence emission was collected through 520 nm (Partec Cyflow) or 525/50 nm (BD LSR II) band pass filters. Resonance Energy Transfer between CFP and YFP was determined by detecting YFP emission through the 520 nm (Partec Cyflow) or 585/42 nm (BD LSR II) band pass filter upon CFP excitation. For AnnexinV-FITC fluorescence detection, FITC was excited with the 488 nm laser and fluorescence emission was detected through a 520 nm band pass filter (Partec Cyflow) or a 525/50 filter (BD LSR II). Propidium iodide was excited with a 488 nm laser (Partec Cyflow) or 561 nm laser (BD LSR II) and fluorescence emission was detected through a 620 nm long pass filter (Partec Cyflow) or a 605/40 band pass filter (BD LSR II).

Fluorescence microscopy and digital imaging

Cell were grown on 22 mm glass bottom dishes (Willco BV, Amsterdam, Netherlands) in 1ml of RPMI-1640 medium supplemented with penicillin (100U/ml), streptomycin (100mg/ml), 10% heat-inactivated fetal bovine serum overnight to let them attach firmly. Cells were pretreated with 100 nM Bortezomib overnight (parental) or for 24 h (HeLa-Bcl-2). Cells were then equilibrated with 30 nM TMRM (Mobitec, Göttingen, Germany) either in 1 ml medium with 100nM Bortezomib, buffered with HEPES (15mM; pH 7.4) and covered with mineral oil (Sigma-Aldrich) or in 2 ml medium with 100nM Bortezomib and fumigated with 5% CO2, and placed on the microscope stage in a heated (37°C) incubator (Solent Scientific, Segensworth, UK). Fluorescence was observed using a Nikon Eclipse TE2000-S microscope equipped with a 40 x numerical aperture 1.3 oil immersion objective, and a dual camera adaptor (Nikon, MicronOptical, Enniscorthy, Ireland). The fluorescence excitation light source was a xenon bulb inside a monochromator with 15nm spectral band width (Polychrome V, Till Photonics, Graefelfing, Germany), set to 435 nm for CFP/FRET, 514nm for YFP, and 550nm for TMRM. The primary beam splitter cube revolver was switched to appropriate transition wave length for CFP (FRET, 458 nm), YFP (520 nm), and TMRM (562nm, all Semrock, Rochester, NY, USA). Emission was detected using a secondary multichroic beamsplitter for CFP/TMRM and YFP (Chroma Bellow Falls, VT, USA) and band path emission filters for CFP 470±24 nm, FRET/YFP 535±30 nm, and TMRM 605±70 nm (Chroma, ET series). Images were taken simultaneously for CFP and FRET and separately for YFP and TMRM every two minutes using two Hamamatsu Orca 285 CCD cameras (Molecular Devices Ltd, Wokingham, UK). The imaging setup was controlled by MetaMorph 7.1.70 software (Molecular Devices Ltd.). IMS-RP fluorescence was observed in the TMRM channel and FRET imaging was performed as described before. mCherry-GFP-LC3 fluorescence was detected using a confocal microscope (LSM 710, Carl Zeiss Jena, Germany) equipped with a 63x 1.4NA oil immersion objective controlled by ZEN 2009 and the Multiple Time Series software. GFP was excited using the 488 nm line of the argon laser and the 488/543 nm multi chroic beam splitter. Emission was detected in the range of 489 to 550 nm. Using the multi track mode to avoid crosstalk of GFP into the mCherry channel, mCherry was excited during the 2nd track by the HeNe laser emitting at 543nm, with the same beam splitter, while the emission was detected in the range of 562 to 727nm. Images were processed and analyzed using ImageJ (Wayne Rasband, NIH, USA) and MetaMorph 7.5 software (Molecular Devices Ltd, Wokingham, UK).

References

1. Kugler W, Buchholz F, Kohler F, Eibl H, Lakomek M, Erdlenbruch B. Downregulation of Apaf-1 and caspase-3 by RNA interference in human glioma cells: consequences for erucylphosphocholine-induced apoptosis. Apoptosis 2005 Oct; 10 (5): 1163-1174.

2. Hashimoto T, Yamauchi L, Hunter T, Kikkawa U, Kamada S. Possible involvement of caspase-7 in cell cycle progression at mitosis. Genes to Cells 2008; 13 (6): 609-621.

3. Afshar G, Jelluma N, Yang X, Basila D, Arvold ND, Karlsson A, et al. Radiation-induced caspase-8 mediates p53-independent apoptosis in glioma cells. Cancer Res 2006 Apr 15; 66 (8): 4223-4232.

4. Imamura R, Konaka K, Matsumoto N, Hasegawa M, Fukui M, Mukaida N, et al. Fas ligand induces cell-autonomous NF-kappaB activation and interleukin-8 production by a mechanism distinct from that of tumor necrosis factor-alpha. J Biol Chem 2004 Nov 5; 279 (45): 46415-46423.

5. Wilkinson JC, Cepero E, Boise LH, Duckett CS. Upstream regulatory role for XIAP in receptor-mediated apoptosis. Mol Cell Biol 2004 Aug; 24 (16): 7003-7014.

6. Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, et al. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 2005 Feb; 25 (3): 1025-1040.