Data for manuscript submitted to ELife and BioArxiv as follows:
CYRI-B mediated macropinocytosis drives metastasis via lysophosphatidic acid receptor uptake

Running title: CYRI-B drives metastasis

Savvas Nikolaou1, Amelie Juin1, Jamie A. Whitelaw1, Nikki R. Paul1, Loic Fort1,2, Colin Nixon1, Heather J. Spence1, Sheila Bryson1 and Laura M. Machesky 1, 3, % 
1. CRUK Beatson Institute, Switchback Road, Bearsden, Glasgow, G61 1BD
2. Present Address: Department of Cell and Developmental Biology, Vanderbilt University, Nashville TN, USA 37240-7935
3. Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1BD
% Author for correspondence laura.machesky@glasgow.ac.uk alternative email lmm202@cam.ac.uk

ORCID:
Savvas Nikolaou: 0000-0001-6936-5911
Amelie Juin: 0000-0002-1481-000X
Jamie Whitelaw: 0000-0001-6739-1032
Loic Fort: 0000-0001-6939-3621
Colin Nixon: 0000-0002-8085-2160
Nikki Paul: 0000-0002-0953-4709
Sheila Bryson: 000-0003-0800-3129
Laura Machesky: 0000-0002-7592-9856

Conflict of interest:
The authors declare that they have no potential conflicts of interest.

Supplementary Figure S1: End-point CKPC tumors show comparable proliferation, apoptosis vascularisation and necrosis to KPC tumours.
A) Representative images of PDAC sections in KPC and CKPC end-point mice, stained for Ki-67 (proliferation). Scale bars, 100µm. Red arrows indicate the positive cells. 
B) Quantification of Ki-67 positive nuclei per area from A. Mean ± SD; Unpaired t-test was performed in n=12 KPC and 12 CKPC independent mice. p-value: ns. 
C) Representative images of PDAC sections in KPC and CKPC end-point mice, stained for cleaved caspase 3 (CC-3, apoptosis). Scale bars, 100µm. Red arrows indicate the positive cells. 
D) Quantification of CC-3 positive cells per area from c. Mean ± SD; Unpaired t-test was performed in n=12 KPC and 12 CKPC independent mice. p-value: ns. 
E) Representative images of PDAC sections in KPC and CKPC end-point mice, stained for CD31 (endothelial marker). Scale bars, 100µm. Red arrows indicate the positive area for CD31. 
F) Quantification of CD31 positive area per tumour area from E. Mean ± SD; Mann-Whitney test was performed in n=12 KPC and 12 CKPC independent mice. p-value: ns = not significant. 
G) Representative images of PDAC section in KPC and CKPC end-point mice, stained for haematoxylin and eosin (H&E) to identify necrotic areas. Scale bar, 500µm. Red arrows indicate the positive area for necrosis. x20 objective used to show the fragmented nuclei within the necrotic areas (Red arrows). Scale bar, 100µm. 
H) Quantification of necrotic area per tumour area from G. Mean ± SD; Mann-Whitney test was performed in n=12 KPC and 12 CKPC independent mice. p-value: ns.

Supplementary Figure S2: Loss of CYRI-B does not alter the formation of PanIN lesions in 10-week old mice
A) Quantification of the number of ducts present in 10-week-old pancreas in KPC and CKPC mice (n≥5 mice). Mean ± SD; unpaired t-test was performed. ns=not significant. 
B) Classification and scoring of pancreatic ducts at in pancreas from 10-week-old KPC and CKPC mice (n≥5 mice). Mean ± SD; unpaired t-test was performed. Ns=not significant.

Supplementary Figure S3: Deletion of CYRI-B in KPC-1 cells does not affect proliferation
A) Representative Western blot from KPC-1 cells for CYRI-B KO. Empty vector was used as control. For CYRI-B KO, Ex3 and Ex4 sequences were used. Alpha-Tubulin was used as loading control. Molecular weights as indicated on the side. 
B) Proliferation assay of control or CYRI-B KO KPC-1 cells from A). 104 cells were seeded on Day1 and manually counted every day. Error bars represent mean ± SEM from n=3 independent repeats. 

Supplementary Figure S4: Loss of CYRI-B results in enhanced spreading and Arp2/3 leading edge recruitment in PDAC cells
A) Western blot images of CKPC-1 cells stably expressing CYRI-B-p17-GFP or GFP. KPC-1 and KPC-2 cell lines were used as control. Untransfected CKPC cells were also used as a control.  Membranes were probed for anti-GFP (bottom blot) and anti-CYRI-B (top blot). GAPDH was used as loading control. Molecular weights are displayed on the side. 

B) Representative immunofluorescence images of CKPC CYRI-B KO and rescued cells. Cells were seeded on fibronectin coated coverslips, fixed and stained for F-actin (magenta), ArpC2 (cyan) and DAPI for nuclei (yellow). Scale bars, 20μm. Yellow dotted boxes show the sites for the magnified images. Red arrows show the positive area for ArpC2 staining at the leading edge. Scale bars, 5μm  Graph shows manual quantification of the number of cells presenting with lamellipodia (purple) or other protrusions (green) from B. Mean ± S.D.; Paired t-test was performed in n=4. p-value as indicated. 
C) Quantification of cell area per cell from B) based on the F-actin staining. Scatter plot here is presented as super plot and every independent biological repeat is coloured differently. Mean ± S.E.M; Paired t-test was performed in n=4. p-value as indicated.
D) Manual quantification of the length of the cell periphery showing strong ArpC2 accumulation at, normalised to the total cell periphery. Scatter plot here is presented as a super plot and every independent biological repeat is coloured differently. Mean ± S.E.M; Unpaired t-test was performed in n=3 (from a total of 30 cells). p-value as indicated.
E) Manual quantification of the relative intensity of ArpC2 on the plasma membrane to cytoplasmic average intensity. Scatter plot here is presented as a super plot and every independent biological repeat is coloured differently. Mean ± S.E.M; Unpaired t-test was performed in n=3. p-value as indicated.

Supplementary Figure S5: Deletion of CYRI-B abolishes chemotaxis

A) Representative spider-plots from n=3 independent chemotaxis assays of KPC-1 control or CYRI-B KO (EX3 and EX4) cells. Cells were seeded on fibronectin coated coverslips and the “Insall” chamber was assembled. A chemotactic gradient of 10% FBS was established and cells were imaged for 16h (1 frame/15min). Every cell trajectory is displayed with a different colour and the displacement of each cell is reported in the x and y-axis. Orange gradient above shows the FBS gradient. Rose-plot data are displayed for each condition below. Red dashed lines show the 95% confidence interval for the mean direction in the Rose plots. The numbers represent degrees of the angle of migration relative to the chemoattractant, with zero (red) denoting the direction of the chemoattractant gradient.
B) Quantification of the results in A) showing the cos(θ) data (chemotactic index). Mean ± S.E.M from the average cos(θ) data of every repeat; One-Way ANOVA followed by Tukey’s multiple comparisons test was performed. Red dashed lines were indicated show the 95% confidence interval for the mean direction in the Rose plots. p-values as indicated on the graph.

Supplementary Figure S6: CYRI-B localises at macropinocytic cups in COS-7 cells 
Still image from live-cell imaging of COS-7 CYRI-B KO cells transfected with CYRI-B-p17-GFP (cyan). 70kDa Dextran was added to the medium to visualise the macropinocytic events (magenta). Scale bar, 5μm. Yellow box shows the magnified area of interest, showing the macropinocytic cups. Scale bar, 5μm. Scatter plot represents the lifetime of CYRI-B+ macropinosomes once internalised. Mean ± SD. Orange box shows a representative montage of CYRI-B internalisation via macropinocytosis. n=15 events from a total of 6 cells.

Supplementary Figure S7: Loss of CYRI-B alters membrane localisation of LPAR1 but not its expression 
A) qPCR analysis for endogenous gene expression of LPAR1 and LPAR3 in CKPC-1 stable cells either transfected with GFP or CYRI-B-GFP. The histogram shows the relative mRNA expression from rescued CYRI-B-GFP and normalised from GAPDH expression. Error bars show the mean ± SD. Unpaired t-test was performed in n=5 independent repeats. p-value as indicated. 
B) Representative immunofluorescence images of CKPC-1 CYRI-B KO and rescued cells. Cells were transfected with LPAR1-HA, then seeded on fibronectin coated coverslips, fixed and stained for F-actin (magenta), anti-HA (cyan) and DAPI for nuclei (yellow). Scale bars, 10μm. Orange dotted boxes show the sites for the magnified images (inset). Red arrows show the positive area for LPAR1 staining at the leading edge. Scale bars, 1μm 
C) Representative immunofluorescence images from B) as an example of how analysis was performed. Yellow dotted lines show the LPAR1 positive area at the periphery of the cells. Right panel shows the manual quantification of the length of LPAR1 in the periphery of the cells, normalised to the total cell length. Scatter plot here is presented as super plots and every independent biological repeat is coloured differently. Mean ± S.E.M; Unpaired t-test was performed in n=3 (from a total of 28 cells). p-value as indicated. Scale bars, 10μm.
Supplementary Video and Table legends:
Supplementary Video 1 (for Figure 5): CYRI-B is localised at internal vesicles and tubules 
Live-cell video of COS-7 CYRI-B KO cells transfected with CYRI-B-p17-GFP (cyan). Time interval, 5sec. Scale bar, 5μm.

Supplementary Video 2 (for Figure 5): CYRI-B is localised at membrane cups 
Live-cell video of COS-7 CYRI-B KO cells transfected with CYRI-B-p17-GFP (cyan). Time interval, 5sec. Scale bar, 2μm.

Supplementary Video 3 (for Figure 6): CYRI-B is localised at macropinocytic cups and internal macropinosomes in PDAC cells
Live-cell video of BSNA cells stably transfected with CYRI-B-p17-GFP (cyan). 70kDa dextran (magenta) was added to the medium as a marker of macropinocytosis. Time interval, 5sec. Scale bar, 5μm.

Supplementary Video 4 (for Supplementary Figure 6): CYRI-B is localised at macropinocytic cups in COS-7 cells
Live-cell video of COS-7 CYRI-B KO cells transfected with CYRI-B-p17-GFP (cyan). 70kDa dextran (magenta) was added to the medium as a marker of macropinocytosis. Time interval, 7sec. Scale bar, 2μm.

Supplementary Video 5 (for Figure 6): CYRI-B is recruited to macropinocytic cups and precedes Rab5 recruitment 
Live-cell video of COS-7 CYRI-B KO cells transfected with CYRI-B-p17-GFP (cyan) and mRFP-Rab5 (magenta). Time interval, 10sec. Scale bar, 5μm.

Supplementary Video 6 (for Figure 7): LPAR1 internalises via macropinocytosis 
Live-cell video of COS-7 cells transfected with LPAR1-GFP (cyan). 70kDa Dextran was added to the medium to visualise the macropinosomes (magenta). Time interval, 6sec. Scale bar, 2μm.

Supplementary Video 7 (for Figure 7): LPAR1 internalises via CYRI-B positive macropinosomes Live-cell video of CKPC-1 cells transfected with CYRI-B-p17-GFP (cyan) and LPAR1-mCherry (magenta). Time interval, 10sec. Scale bar, 5μm.