Inhibition of cell migration and invasion mediated by the TAT-RasGAP317–326 peptide requires the DLC1 tumor

15 min

30 min

45 min

60 min

TAT

TAT-Ras

GAP

317-326

Control

TAT

TAT-Ras

GAP

317-326

Control

15 min

30 min

45 min

60 min

Trypsin-mediated detachment

Adhesion rate

B

480 min

480 min

C

TAT TAT-RasGAP317-326 Control

Cell spreading 30 min after plating

Figure S1

Figure S1. Kinetics of TAT-RasGAP317-326-mediated adhesiveness increase.

A. U2OS cells were subjected to an adhesion assay for 30 minutes in presence of the indicated concentrations of TAT and TAT-RasGAP317-326 (the statistical significance of the observed

differences was assessed by one-way ANOVA).

B. Representative images of the adhesion assay performed in Figure 1E. Scale bar: 100 µm. C. Higher magnification of cells 30 minutes after plating. Scale bar: 40 µm.

A

0 200 400 500 300 0 5 10 15 20 Peptide concentration [µM] TAT TAT-RasGAP317-326

Number of cells per mm

2 100

*

*

*

*

: TAT vs. TAT-RasGAP317-326

0 75 50 100 M21 M21L % adherent cells

Control w/o trypsin Control with trypsin

TAT-RasGAP_317-326 with trypsin

TAT with trypsin

25 0.93 0.32 +/ -0.44 0.32 +/ -0.15 0.02 +/ -0.41 0.27 +/ -integrin-α_V +

-B

integrin-β₁ 1.13.105 integrin-α3 4.45.104 integrin-αV 2.72.104 integrin-β5 3.00.104 integrin-α2 1.34.104 integrin-α6 7.38.103 integrin-α5 2.97.103 integrin-β2 < 5.102 integrin-α₉ < 5.102 < 5.102 integrin-β₄ < 5.102 integrin-α₄ integrin-β₃ N/A integrin-β_6-8 integrin-α1 integrin-α7-11 N/A N/A N/A Copies/cell Protein

D

48h 24h 48h 72h -48h siGFP si-Integrin-β1 130 130 kDa HeLa 0.11 0.11 +/ -0.10 0.20 +/ -0.24 0.26 +/ -0.16 0.10 +/ -0 75 50 100 25 % adherent cells

siGFP siIntegrin-β1HeLa

Integrin-β1 RasGAP

C

A

α₁β1 α₂β1 α₃β1 α₄β1 α₅β1 α₆β1 α₇β1 α₈β1 α₉β1 α₁₀β1 α₁₁β1 α_Vβ1 α_Xβ2 α_Mβ2 α_Lβ2 α_Dβ2 α_Vβ3 α6β4 αVβ5 αVβ6 αEβ7 αVβ8 α₄β7 β2 β1 β3 β4 β5 β6 β7 β8 α₂β3 α integrin subunits 48h- 48h 24h 48h 72h siGFP siCD44 130 90 kDa CD44 RasGAP HeLa 1.3 0.6 +/ -5.0 1.9 +/ -1.7 2.3 +/ -3.0 1.1 +/ -0 75 50 100 25 % adherent cells

siGFP siCD44HeLa

E

F

0 200 400 600

PBS Collagen Fibronectin Laminin

1.5 +/ 3.3 -0.0 +/ 0.0 -0.0 +/ 0.0 -0.0 +/ 0.0 -4.5 +/ 4.1 -0.6 +/ 0.6 -0.0 +/ 0.0 -0.0 +/ 0.0 -0.3 +/ 0.6 -0.0 +/ 0.0

-Number of cells per mm

2

Control w/o trypsin

Control with trypsin TAT-RasGAPTAT with trypsin_317-326 with trypsin

PBS Collagen Fibronectin Laminin

TAT TAT-Ras

GAP317-326

Control w/o trypsin Control

With trypsin

Figure S4

Figure S4. Integrins, CD44 and caspases are not required for TAT-RasGAP317-326-mediated adhesion increase. A. The twenty-four known integrin heterodimers are represented (8).

B. Wild-type (M21) or αV-integrin-null (M21L) melanoma cells were treated 8 hours with 20 µM TAT, 20 µM TAT-RasGAP317-326 or left untreated. The cells

were then subjected to a trypsin-mediated detachment assay.

C. HeLa cells were transfected with siRNAs targeted against GFP (siGFP) or β1-integrin (siIntegrin-β1). Forty-eight hours later, the cells were incubated 8 hours with 20 µM TAT, 20 µM TAT-RasGAP317-326 or left untreated and were subjected to a trypsin-mediated detachment assay. The upper panel depicts the

loss of β1-integrin expression after siRNA transfection.

D. Expression levels of the indicated integrin family members in U2OS cells [data taken from (9)].

E. CD44 was silenced in HeLa cells with siRNAs. Forty-eight hours later, the cells were incubated 8 hours with 20 µM TAT, 20 µM TAT-RasGAP317-326 or left

untreated. The cells were then subjected to a trypsin-mediated detachment assay. The upper panel depicts the loss of CD44 expression after siRNA transfection.

F. U2OS cells were grown overnight on bacteriological Petri dishes that had been coated beforehand with collagen, fibronectin or laminin. The cells were then treated or not with 20 µM TAT or 20 µM TAT-RasGAP317-326 for 8 hours. A trypsin-based detachment assay was finally performed. Representative