How To Understand Cell Biology

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Rosanna Thornton
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1 Supplemental Material Goldoni et al., THE JOURNAL OF CELL BIOLOGY Figure S1. Specific high affinity interaction between decorin and Met-Fc. (A) Ligand-binding assays using HGF as soluble ligand and Met-FC as immobilized substrate. (B) Ligand-binding assays using either decorin core or mouse Ig as soluble ligands and Met-Fc as immobilized substrate. (C) Ligand-binding assay using perlecan s LG3 module as soluble ligand and Met-Fc as immobilized substrate. LG3 is also His tagged as the decorin protein core. Data represent mean ± SEM of three independent experiments performed in triplicate. 1

(B) Ligand-binding assays using either decorin core or mouse Ig as soluble ligands and Met-Fc as immobilized substrate.

2 Figure S2. Decorin induces shedding and internalization of the Met receptor. (A) Slot blot of HeLa-conditioned media probed with an antibody raised against the ECD of the Met receptor. Cells were treated with 100 nm decorin for the times indicated. (B, top) Representative Met immunoblot of HeLa cells treated with 100 nm decorin for 30 min in the presence or absence of TIMP-2 or TIMP-3 (both 1 µm). Cells were preincubated with the inhibitors for 30 min. (bottom) Quantification of immunoblots as shown in the top panel (*, P < 0.05). Error bars indicate SEM. (C) Slot blot of HeLa-conditioned media from the experiment shown in B probed with an antibody raised against the ECD of the Met receptor. Note that the control medium in panel C was conditioned for 24 h, whereas in A it was conditioned for the time points indicated. Media from the decorin treatment in the presence or absence of the inhibitors were changed and collected after 1-h incubation. (D, top) Fluorescence images of the Met receptor cellular localization after 100 nm decorin treatment for the time indicated. A Met antibody recognizing the receptor ECD was used. (bottom) Confocal images recorded from the same experiment shown in the top panel. Arrows indicate internalized Met. Bars, 10 µm. (E) Representative immunoblot of Met receptor levels in HeLa cells after 100 nm decorin treatment followed by trypsin digestion. EGFR was used as control to validate this approach. GAPDH was used as loading control. S2

(B, top) Representative Met immunoblot of HeLa cells treated with 100 nm decorin for 30 min in the presence or absence of TIMP-2 or TIMP-3 (both 1 µm).

3 Figure S3. Decorin induces apoptosis via Met receptor signaling. (A) Immunoblot of HeLa cells showing PARP cleavage after 300 nm decorin treatment for 24 h in the presence or absence of 1 µm SU Actin was used as loading control. (B) Caspase 3/7 activity was measured using the Caspase-Glo 3/7 assay (Promega). HeLa cells were incubated with 300 nm decorin for 24 h as in A in the presence or absence of 40 µm VAD, a pancaspase inhibitor, and 1 µm SU11274 as indicated. 100 µm etoposide was used as positive control. The values represent the mean ± SEM of three independent experiments performed in triplicate. (C) Representative immunoblot showing Met down-regulation after 30 min of 100 nm decorin treatment in full-serum medium. After removing decorin and replacing the medium for the time indicated, Met expression recovers. RLU, relative light units. S3

HeLa cells were incubated with 300 nm decorin for 24 h as in A in the presence or absence of 40 µm VAD, a pancaspase inhibitor, and 1 µm SU11274 as indicated.

4 Figure S4. Decorin inhibits cell motility by a mechanism that involves both Met and EGFR. (top) Motility assay performed with a monolayer of HeLa cells scratched to cause a wound. Cells were treated for 24 h with 100 nm decorin, 1 µm AG1478, a specific EGFR tyrosine kinase inhibitor, 2 µg/ml H9786, a Met-blocking antibody, or various combinations as indicated. Representative photos are shown. Bar, 40 µm. (bottom) Quantification of three wound closure assays. The same wound area was followed over time. Quantification was performed using ImageJ by averaging three measurements for each wound. Each assay was run in triplicate. Values represent the mean ± SEM (**, P < 0.01). S4

Cells were treated for 24 h with 100 nm decorin, 1 µm AG1478, a specific EGFR tyrosine kinase inhibitor, 2 µg/ml H9786, a Met-blocking antibody, or various combinations

5 Figure S5. 3D structure of the leucine-rich repeats of internalin B and conservation of key amino acid residues within its concave face. (A) Ribbon diagram of the leucine-rich repeats from Listeria monocytogenes internalin B (PDB 1H6T) in which various key aromatic amino acids (red) have been replaced with Ser, resulting in failure to bind to the Met receptor. The structure was rendered with PyMol program. (B) Alignment of amino acid sequences for human and bovine decorin with internalin B using the Clustal W algorithm. Hydrophilic and hydrophobic residues are shown in blue and red, respectively. The vertical arrow denotes the conserved Y170. 5

replaced with Ser, resulting in failure to bind to the Met receptor. The structure was rendered with PyMol program.

Supplemental Fig. S1. The schematic diagrams of the expression constructs used in this study.

1 Supplemental data Supplemental Fig. S1. The schematic diagrams of the expression constructs used in this study. Supplemental Fig. S2. Ingenuity Pathway Analysis (IPA) of the 56 putative caspase substrates