Supplemental Fig. S1 Svasn Interacts with Tgfbeta1

Supplemental Data.

Supplemental Fig. S1 sVASN interacts with TGFbeta1

CHO cells permanently transfected with VASN were treated during three hours without or with 10 pM of TGF and without or with 10 µM of BB-94 as indicated. Then, the conditioned media was harvested and treated with antibodies against VASN. Immune complexes were collected with protein A, washed and analyzed by ELISA to detect TFGbeta1 (A) or analyzed by Western blot with antibodies against VASN (B). A, The results of two independent experiments were quantified and the results expressed as averages. Note that BB-94 prevents the generation of soluble VASN (B) and, therefore, the conditioned medium of cells treated with the metalloprotease inhibitor was used as a negative control.

Supplemental Fig. S2 Treatment of CHO with the ADAM17 inhibitor BB-94 upregulates TGF signaling

Wild type CHO cells were treated during three hours with different concentrations of TGF and with or without 10 µM of BB-94 as indicated. Then, cells were lysed and cell lysates analyzed by Western blot with antibodies against pSmad2 or total Smad2 as described under experimental procedures. The results of four independent experiments were quantified and the results expressed as averages + S.D.

Supplemental Fig. S3. The metalloprotease inhibitor BB94 prevents the cleavage of VASNand enhances autocrine TGFbeta signaling

BT474 cells were cultured without serum and without or with 10 µM of BB-94 for 48 hours. Then, the conditioned media was analyzed by Western blot with antibodies against the extracellular domain of VASN. The cells were lysed and cell lysates analyzed by Western blot with antibodies against pSmad2 or total Smad2 as described under experimental procedures. The results of two independent experiments were quantified and the results expressed as averages.

Supplemental Fig. S4. The metalloprotease inhibitor BB94 sensitizes cells to the inhibition of proliferation induced by TGFbeta .

MDA-MB-231 cells were treated without or with different concentrations of TGF-beta and with or without 10 µM of BB-94 in serum-free medium during four days. Then cells were counted.The results of two independent are expressed as averages.

Supplemental Fig. S5. The metalloprotease inhibitor BB94 does not affect BMP4 signaling.

MCF7 cells were treated without or with different concentrations of BMP4 and with or without 10 µM of BB-94 in serum-free medium during 5 hours. Then, the cells were lysed and cell lysates analyzed by Western blot with antibodies against pSmad1,5,8 or total Smad1,5,8 as described under experimental procedures. The results of two independent experiments were quantified and the results expressed as averages.

.

Supplemental Fig. S2. Analysis of the shedding of VASN in the M2 mutant cell line.

Cell lysates or conditioned media from wild type (WT) CHO cells or CHO cell mutants transfected with empty vector (M2) or the same vector expressing wild type ADAM17 (M2/A17), were analyzed by Western blot with an antibody against the extracellular domain of VASN.Ponceau S staining was used as loading control. The results of three independent analyses were quantified. The results were expressed as percentages relative to soluble VASN in WT CHO cells and are represented as averages + S. D.