AMBRA1 Is Able to Induce Mitophagy Via LC3 Binding

Supplementary Figure Legends

Supplementary Figure 1: Endogenous AMBRA1 binds LC3 following CCCP treatment in the mitochondrial fraction

A) Endogenous AMBRA1 binds LC3 following FCCP treatment. Mitochondrial extracts from

HEK293 cells were immunoprecipitated using an anti-AMBRA1 antibody. Purified complexes and corresponding total extracts were analysed by Western Blot (WB), using anti-AMBRA1 and anti-

LC3 antibodies.

B) AMBRA1 does not bind LC3 in total extracts in both normal and starvation conditions.

Total extracts from HEK293 cells transfected with a vector encoding myc-AMBRA1-WT were immunoprecipitated using an anti-myc antibody. Purified complexes and corresponding total extracts (normal condition or starvation condition) were analysed by Western Blot (WB), using anti-AMBRA1 and anti-LC3 antibodies.

Supplementary Figure 2: Bcl-2-ActA co-localizes with the mitochondrial network, without inducing mitochondria aggregation

HEK293 cells were transfected with a vector encoding Bcl-2-ActA. Twenty-four hours after transfection, cells were fixed and stained with an anti-Bcl-2 antibody (green). Nuclei were stained with DAPI 1µg/µl for 20 min. Merge of the fluorescence signals is shown. N, Nucleus. Scale bar,

Suppl Figure 3: Dynamics of mitochondrial shape changes following AMBRA1-ActA overexpression

HEK293 cells were co-transfected with vectors encoding myc-AMBRA1-ActA and mito-RFP. 8,

10 or 18 hours after transfection, cells were fixed and stained with an anti-myc antibody (green) Ambra1 regulates mitophagy

Merge of the two fluorescence signals is shown in the right panel. The insert within the 10h time point panel (i) shows AMBRA1-ActA rings (green) around mitochondria (red). N, nucleus; Mito- agg., Mito-aggresomes. Scale bar, 6µm (Scale bar in the insert, 400nm).

Supplementary Figure 4: AMBRA1-ActA co-localizes on mitochondria with ubiquitin, p62 and LC3 in HEK293 cells

HEK293 cells were co-transfected with vectors encoding myc-AMBRA1-ActA and mito-RFP (red).

Twenty-four hours after transfection, cells were fixed and stained with anti-myc (blue), anti-

Ubiquitin, anti-p62 or anti-LC3 (green) antibodies. Merge of the different fluorescence signals are shown. Scale bar, 5µm. Quantification of co-localizations is shown by the graph.

Supplementary Figure 5: AMBRA1WT does not induce mitochondria redistribution around the nucleus

Ultrastructural analysis by EM on whole cells. HEK293 cells were co-transfected with vectors encoding myc-AMBRA1WTand GFP-mitochondria. Twenty-four hours after transfection, GFP- positive cells were sorted by using FACS and fixed in glutaraldehyde. Morphological analysis was performed. Arrows indicate normal mitochondria. Scale bar, 5µm.

Supplementary Figure 6: AMBRA1-ActA induced mitophagy is dependent of Pi3PK-complex

A) Wortmannin treatment partially inhibits AMBRA1-ActA-mito-RFP co-localization on mito- aggresomes. HEK293 cells were co-transfected with vectors encoding AMBRA1-ActA and mito-

RFP and directly treated with 75nM of Wortmannin. After 24 hours, cells were fixed and stained with an anti-myc antibody (green). Nuclei were stained with DAPI 1µg/µl for 20 min. Merge of the three fluorescence signals is shown in the right panel. N, Nucleus. Scale bar, 8µm. B) Ambra1-

ActA co-localizes on mitochondria with LC3 and LAMP1. HEK293 cells were transfected with

2 Ambra1 regulates mitophagy vectors encoding myc-AMBRA1-ActA and mito-RFP. Twenty-four hours after transfection, cells were treated 1h with chloroquine, fixed and stained with anti-LC3 (green) and anti-Lamp1 (blue) antibodies. The merge of the fluorescence signals are shown. Scale bar, 8µm. C) Down-regulation of BECLIN 1 delays AMBRA1-ActA-induced mitophagy in HEK293 cells. HEK293 cells were transfected with a control SiRNA (Ctrl) or a SiRNA directed against BECLIN 1. Twenty-four hours after downregulation of BECLIN 1, cells were transfected with a vector encoding myc-AMBRA1-

ActA. Twenty-four, 48 and 72h after transfection, protein extracts were analysed by western-blot using the following antibodies: anti-MnSOD, anti-TOM20, anti-myc (to control AMBRA1-ActA expression), and anti-ACTIN (loading control). The graph represents the MnSOD/ACTIN ratio

(± SD). Each point value represents the mean ± SD from three independent experiments. Statistical analysis was performed using Student Test (*P < 0.05) 24h versus 72h.

D) HEK293 cells were transfected with a control SiRNA (Ctrl) or a SiRNA directed against

BECLIN 1. Twenty-four hours after downregulation of BECLIN 1, cells were co-transfected with vectors encoding myc-AMBRA1-ActA and mito-RFP. Twenty-four, 48 and 72 hours after transfection, cells were fixed and stained with anti-myc antibody (green) and anti-BECLIN 1 (blu).

Merge of the three fluorescence signals is shown in the right panel. Scale bar, 6μm. The graph shows the quantification of mito-RFP dots per cell positive for AMBRA1-ActA (± SD). Each point value represents the mean ± SD from three independent experiments. Statistical analysis was performed using Student Test (*P < 0.05).

Supplementary Figure 7: AMBRA1-ActA induces mitochondrial clearance in a proneural cell line ETNA cells were co-transfected with vectors encoding myc-AMBRA1-ActA and mito-RFP. 24, 48 and 72 hours after transfection, cells were fixed and stained with an anti-LC3 antibody (green).

Merge of the two fluorescence signals is shown in the right panel. Scale bar, 6μm The graph shows the quantification of AMBRA1-ActA-mito-RFP co-localization on mito-aggresomes per cell (±

3 Ambra1 regulates mitophagy

SD). Each point value represents the mean ± SD from three independent experiments. Statistical analysis was performed using Student Test (*P < 0.05), 24h versus 72h.

Supplementary Figure 8: AMBRA1-ActA induces translocation of PARKIN to mitochondria

A) HEK293 cells were transfected with vectors encoding myc-AMBRA-ActA and mito-RFP (red).

Twenty-four hours after transfection, cells were fixed and stained with anti-myc (AMBRA1) and anti-PARKIN (green) antibodies. Merge of the fluorescence signals are shown. Scale bar, 8µm. B)

HEK293 cells were transfected with vectors encoding myc-AMBRA1WTand mito-RFP (red).

Twenty-four hours after transfection, cells were fixed and stained with anti-myc (AMBRA1) and anti-PARKIN (green) antibodies. The merge of the fluorescence signals is shown. N, Nucleus.

Scale bar, 8µm.

Supplementary Figure 9: AMBRA1-ActA co-localizes on mitochondria with Ubiquitin, LC3 but not with p62 in HeLa cells

HeLa cells were co-transfected with vectors encoding myc-AMBRA1-ActA and mito-RFP (red).

Twenty-four hours after transfection, cells were fixed and stained with anti-myc (blue), anti-

Ubiquitin, anti-p62 or anti-LC3 (green) antibodies. Merge of the different fluorescence signals are shown. Scale bar, 5µm. Quantification of co-localization is shown by the graph.

Supplementary Figure 10: Analysis of mitochondrial depolarization upon AMBRA1-ActA expression.

A) AMBRA1-ActA surrounds depolarised mitochondria. HeLa cells were co-transfected with vectors encoding myc-AMBRA1-ActA and GFP-mitochondria. To be sure that almost all green cells were positive for AMBRA1-ActA, we transfected DNA following the ratio 3 (GFP-mito):1

(AMBRA1-ActA). Twenty-four hours after transfection, the cell medium was changed with HBSS

4 Ambra1 regulates mitophagy medium (free of phenol) and cells were loaded 30 min with 50 nm TMRM. Cells were exposed or not to FCCP (1μM), in order to induce strong mitochondrial depolarization as a control. Cells were then observed in live imaging under 60X immersion oil on a Zeiss LM 700 confocal microscope.

Quantification of TMRM was performed using ImageJ software. The graph illustrates quantification

TMRM value (± SD). Each point value represents the mean ± SD from three independent experiments. Statistical analysis was performed using Student Test (*P < 0.05), untransfected cells versus AMBRA1-ActA-positive cells. N, Nucleus. Scale bar, 5µm. B) HeLa cells were transfected with a vectors encoding GFP-mitochondria. Twenty-four hours after transfection, the cell medium was changed with HBSS medium (free of phenol) and cells were loaded 30 min with 50nm TMRM.

Cells were then observed in live imaging under 60X immersion oil on a Zeiss LM 700 confocal microscope. Scale bar, 10µm.