JCS046250 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Figure S1. Expression profile of Atg4 and Atg8 paralogues in human cell-lines. RT-PCR reactions for controls (Actin and GAPDH) (A), Atg4A-D (B) and the Atg8 paralogues LC3, GATE-16 and GABARAP-L1 (C) using cDNA obtained from A431, HeLa and MCF-7 cell-lines.

• Supplemental Figure S2 -

  Figure S2. Localisation of Atg4D in human cells. Top panels show a HeLa cell transiently expressing GFP-Atg4D, following fixation and labelling with anti-pericentrin antibodies. GFP-Atg4D is cytoplasmic with a strong enrichment at the centosome (arrow). Bottom panels show HeLa cells labelling with anti-Atg4D and anti-pericentrin antisera. A similar pattern of Atg4D distribution including strong centrosomal enrichment (arrows) is seen. Scale bars: 10m.

• Supplemental Figure S3 -

  Figure S3. Examples fields of HEK293 cells stably expressing YFP-LC3 and HeLa cells stably expressing GABARAP-L1, transiently expressing CFP, CFP-Atg4B, CFP-Atg4D and CFP-ΔN63 Atg4D. Scale bar: 10µm.

• Supplemental Figure S4 -

  Figure S4. Unlipidated GABARAP-L1 (GP-L1-I) is enriched in membranes and is resistant to salt washing. Membranes were isolated from GFP-LC3 and GFP-GABARAP-L1 stable cells and were incubated in acetate sucrose buffer (A/S) or in A/S containing 1% Triton X-100 (TX-100), 500 mM NaCl 500 mM KCL or 100 mM NaCO₃ pH 11. Membrane pellets and wash fractions were immunoblotted using GFP antibodies and with anti-calnexin antisera.

• Supplemental Figure S5 -

  Figure S5. Example images of cell death assessed in Fig. 6E,F. Arrows depict pyknotic chromatin. Scale bars: 10 µm.

• Supplemental Figure S6 -

  Figure S6. ΔN63 Atg4D translocates to mitochondria as a prelude to cell death. Frames from a live-cell imaging experiment of an untreated A431 cell transiently expressing GFP-ΔN63 Atg4D and Mito-dsRed. Scale bar: 10 µm.

• Movie 1 -

  Movie 1. Transient recruitment of Atg4D to mitochondria as a prelude to cell death. An A431 cell transiently co-expressing GFP-Atg4D (top panel, green) and mito-dsRed (middle panel, red). About 20 minutes before cell rounding and apoptosis, GFP-Atg4D rapidly and transiently associates with mitochondria (yellow signal, merged panel).

• Movie 2 -

  Movie 2. Caspases are not required for recruitment of Atg4D to mitochondria. An A431 cells transiently co-expressing GFP-Atg4D and mito-dsRed undergoing apoptosis in the presence of zVAD.FMK.

• Movie 3 -

  Movie 3. Atg4D is recruited to mitochondria during staurosporine-induced apoptosis. An A431 cell transiently co-expressing GFP-Atg4D and mito-dsRed undergoing apoptosis in the presence of staurosporine. Arrows depict mitochondrial Atg4D recruitment.