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1 DOI: /ncb2386 Figure 1 Src-containing puncta are not focal adhesions, podosomes or endosomes. (a) FAK-/- were stained with anti-py416 Src (green) and either (in red) the focal adhesion protein paxillin, the adhesion/podosome protein cortactin, Rab5 as a marker of early endosomes or Rab7 as a marker of late endosomes. Arrows indicate lack of co-localisation in puncta. Scale bars: 20mm. (b) FAK -/- cells were fixed in paraformaldehyde and gluteraldehyde and the pellets embedded in LR-white resin. Ultrathin sections were cut and then stained using anti-py416-src antibody which was detected using gold-conjugated protein A.Sections were stained with uranyl acetate and lead citrate prior to imaging using an electron microscope. Images show active Src sequestered in interior of single-membrane delimited vesicles characteristic of intermediate stages of the autophagy pathway. Solid arrows indicate 10nm gold particles. Scale bars: 0.2mm. (c) Single colour images are shown of FAK +/+ and -/- cells stained with anti-py416 Src (red) and LC3B, Atg7 or Atg12 (green). Scale bars: 20mm. (d) FAK +/+ cells were stained with Atg7 (green) and either anti-fak or paxillin (red). Merged images are shown. Solid arrows indicate co-localisation. Scale bars: 20mm. 1

2 Figure S2 Src localises to Atg7 puncta in tumour tissue in vivo in the absence of FAK. (a) Immunohistochemical analysis of FAK in normal pancreatic ducts from FAK expressing (left panel) and FAK deficient (right panel) mice. (b) Immunofluorescence was carried out on tumours collected from FAK expressing and FAK deficient mice using anti-py416-src (green). Solid arrows show active Src at cell periphery while broken arrows show active Src in puncta. Quantification is also shown. Data is presented as mean ± s.d. and significance is p < (n = 3). (c) Immunofluorescence on FAK deficient tumours is shown using anti-py416-src (red) and anti-atg7 (green). Dotted arrows indicate co-localisation in puncta. Scale bars: 20µm. 2

3 Figure S3 Autophagy inhibition reduces localisation of active Src in internal puncta. (a) Cells were untreated or treated for 48 hours with 10mM 3MA then fixed and stained with anti-py416-src (green in upper panels) or with anti-py416-src and anti-paxillin antibodies (green and red respectively in lower panels). Solid arrows show co-localisation and broken arrows indicate lack of co-localisation. Scale bars: 20mm. Quantification of the percentage of cells where active Src is internalised is shown. Data is presented as mean ± s.d. and significance is p < (n = 3). (b) Quantification of the percentage of cells expressing scrambled, Atg5 or Atg12 sirna with Src in puncta is shown. Data is presented as mean ± s.d. and significance is p < 0.01 for sirna treated FAK -/- cells (n = 3). (c) Lysates from FAK -/- cells expressing scrambled, Atg5 or Atg12 sirna were immunoblotted with anti-lc3b and anti-actin antibodies. Cells were treated with CHL for 24 hours and densitometry carried out to calculate the amount of LC3B-II relative to actin. Densitometry is presented as a percentage increase for the immunoblot shown. 3

4 Figure S4 RFP-GFP-LC3 positive puncta are affected by autophagy/ lysosomal inhibitors. (a) Cells were transiently transfected with an RFP- GFP tandem fluorescent-tagged LC3 (RFP-GFP-LC3) and treated with 10mM 3MA or 10mM CHL for 24 hours prior to fixation. Scale bars: 20µm. The number of yellow puncta and the number of RFP LC3 positive puncta in the merged images were counted and the total number of puncta per cell calculated. Data is presented as mean ± s.d. (n = 3). (b) FAK -/- cells were transiently transfected with Src-Y527F-GFP or with Src-251-GFP (green) and stained with anti-lc3b (red). Single colour images are shown. Scale bars: 20µm. 4

5 Figure S5 FAK phosphorylation is a key determinant of autophagic targeting. (a) Lysates from FAK -/- cells stably re-expressing FAK-wt, FAK-Y397F or FAK-Y4F-Y9F were immunoblotted with anti-fak, anti-fak-y397, anti- FAK-Y861, anti-fak-y925, anti-fak-y576/577 and anti-actin antibodies. (b) Single color images of FAK-wt, FAK-Y397F or FAK-Y4F-Y9F stained with anti-fak (red) and with anti-py416 Src (green). Scale bars: 20µm. (c) Quantification of the percentage of cells where active Src localized to autophagosomes in cells expressing FAK mutants is shown. Data is presented as mean ± s.d. and significance is p < (n = 3). (d) Cells were stained with anti-py416-src (red) and either LC3B (upper panels) or Atg7 (lower panels). Scale bars: 20µm. (e) The number of yellow puncta and the number of RFP LC3 positive puncta were counted in FAK-wt, FAK-Y397F or FAK-Y4F-Y9F cells and the total number of puncta per cell calculated. Data is presented as mean ± s.d. (n = 3). 5

6 Figure S6 Inhibition of autophagy results in cell death in the absence of FAK. (a) FAK -/- cells treated with 3MA (for 48 hours), dasatinib or CHL (both for 24 hours), or transfected with scrambled, Atg5 or c-cbl sirna were stained with TUNEL (green) and DAPI (blue). Lysates from FAK -/- cells transfected with scrambled or c-cbl sirna were immunoblotted with anti-parp and anti-actin. (b) FAK +/+ cells treated with CHL or transfected with scrambled, Atg5 or c-cbl sirna were stained with TUNEL (green) and DAPI (blue). Quantification is also shown. Data is presented as mean ± s.d. (n = 3). Lysates from FAK +/+ cells transfected with scrambled, Atg5 or c-cbl sirna were immunoblotted with anti-parp and anti-actin. (c) FAK -/- cells expressing c-cbl-ha were transfected with scrambled sirna, c-cbl sirna pool or two individual c-cbl sirna sequences then TUNEL stained. Quantification of percentage of cells positive for TUNEL staining is shown and data is presented as mean ± s.d. (n = 3). Lysates from these cells were then immunoblotted using an anti- PARP antibody (lower right panel). 6

7 Figure S7 p62 and NBR1 do not appear to be involved in localisation of Src to puncta. (a) FAK +/+ and -/- cells were washed and maintained in Earls Balanced Salt Solution (EBSS) for 2 hours. Lysates were then immunoblotted with anti-lc3b and anti-actin antibodies. (b) Lysates from FAK +/+ and -/- cells were immunoblotted with anti-p62 and anti-actin antibodies. (c) FAK -/- cells were fixed and stained for anti-py416-src (green) and p62 (red). Merged and zoomed images are shown. Scale bars: 20µm. (d) Lysates from FAK -/- cells expressing scrambled or NBR1 sirna were immunoblotted with anti-nbr1 and anti-actin antibodies (left panels). Cells were also stained with anti-py416-src (green) and DAPI (blue) (upper right panels) Scale bars: 20µm. The percentage of cells with Src internalised in puncta was quantified and data presented as mean ± s.d. and significance is p > 0.5 (n = 3). 7

8 Figure S8 Model for role of c-cbl in autophagic targeting and degradation of active Src. This model depicts the central role of c-cbl in directing the mode of Src turnover relating to the status of adhesion signalling through the integrin-fak-src pathway. (a) Src, FAK and c-cbl co-localize at focal adhesions in adherent SCC cancer cells. There is evidence that Atg proteins, including LC3, Atg7 and Atg12 are also present at focal adhesions and in intracellular puncta. Under these conditions of normal flux through the pathway, Src turnover is largely via the proteasome, that published work suggests is mediated by ubiquitination of Src by the E3 ubiquitin ligase activity of the Src interacting protein c-cbl. Flux through the integrin- FAK-Src pathway leads to multiple, well described, signalling outputs, which in turn produce the biological consequences of pathway activity, including cell survival. (b) When flux through the integrin-fak-src pathway is blocked, either because FAK is absent, or signalling to FAK from integrins or Src is severely compromised (by phosphorylation site mutation or by cell detachment/anchorage-independent growth of FAK-proficient cells), then active Src is targeted to puncta that also contain multiple autophagy regulators. Under these conditions, active Src is degraded in a lysosomedependent manner that is required for cancer cell survival. Mechanistically, the absence of FAK (or loss of flux) triggers formation of a Src/LC3 complex associated with Src s autophagic targeting which requires c-cbl. Importantly, we have identified an LIR motif in c-cbl that mediates binding to LC3, and which is required for the targeting of Src to the autophagy pathway. Hence, c-cbl is intimately involved in mediating Src turnover; under normal conditions this involves c-cbl E3 ligase activity in directing active Src to the proteasome, while under conditions when FAK is severely perturbed, this involves c-cbl s adaptor function. We conclude that active Src is selectively targeted to the autophagy pathway upon perturbation of its binding partner protein, FAK, which normally tethers it to spatiallydetermined focal adhesion complexes. Autophagic targeting is mediated by a c-cbl-regulated Src/LC3 complex, resulting in lysosomal degradation of active Src, and this is required for cancer cell survival. 8

9 Un-cropped Western blots Figure 1c - Lysates FAK PY416 actin Src Yes Fyn Figure 2c - LC3B IP Lysates Figure 2d - Lysates Src PY416 LC3B Src PY416 LC3B Figure 3b - Lysates Atg5 Atg12 actin actin PY416 Src LC3B actin Figure S9 Uncropped western blots 9

10 Figure 3c - Lysates PY416 Src p53 actin Figure 4a - Lysates Figure 4c - Lysates Src PY416 LC3B actin Figure 4e - Lysates FAK actin LC3B actin LC3B actin Figure 5a - Lysates 397 FAK PY416 Src Figure S9 continued 10

11 Figure 5c LC3B IP Lysates LC3B IP Lysates Src LC3B LC3B 397 actin Figure 6d - Lysates Figure 7b c-cbl IPs Lysates c-cbl IPs Lysates c-cbl IPs Lysates PARP PARP PY416 Src c-cbl Figure 7c - Lysates Figure 7e Figure 8c LC3B IP Lysates LC3B IP Lysates HA c-cbl actin Src LC3B Ponceau Figure S9 continued 11

12 Supplementary Figure 3c - Lysates LC3B actin LC3B actin Supplementary Figure 5a - Lysates FAK actin Supplementary Figure 6a Supplementary Figure 6b Supplementary Figure 6c PARP actin PARP actin PARP actin Supplementary Figure 7a Supplementary Figure 7b Supplementary Figure 7d LC3B actin p62 actin NBR1 actin Figure S9 continued 12