Syndecan-1 regulates _vß₅ integrin activity in B82L fibroblasts

JCS02970 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. FN induces complete spreading of B82L fibroblasts. B82L cells were plated on wells co-coated with increasing amounts of FN and 60 mg/ml mAb 281.2, 150 μg/ml S4ED pAb or in the absence of antibody. Cells were allowed to spread 2 hours before fixation and labeling with Rhodamine-conjugated phalloidin. Bar, 50 μm.

• Supplemental Figure 2 -

  Fig. S2. Competition of B82L cell spreading by RGD-containing peptides. (A) B82L cells were pretreated with 30 μM GRGDSP or 30 μM GRADSP for 10 minutes and plated on 5 μg/ml VN or 60 mg/ml mAb 281.2 plus 1 mg/ml VN for 2 hours before fixing and staining. (B) Relative cell spreading after treatment with a_v-integrin-specific cyclic-RGD-containing peptides (3 μM) is quantified for B82L-hS1 cells plated on substrata coated with 5 mg/ml VN or 60 μg/ml FN, or 60 μg/ml human Sdc1-specific mAb B-B4 in combination with either 1 μg/ml VN or 3 μg/ml FN. Identical results were obtained plating on mAb 281.2 to engage the mouse Sdc1. Bar, 50 μm.

• Supplemental Figure 3 -

  Fig. S3. siRNA blockade of b₅ subunit expression blocks syndecan-induced spreading on FN. B82L cells were plated on wells coated with 60 μg/ml mAb 281.2 and increasing amounts of FN after treatment with β₅-integrin-specific or control siRNA. Cells were allowed to spread 2 hours before fixation and labeling with Rhodamine-conjugated phalloidin. Bar, 50 μm.

• Supplemental Figure 4 -

  Fig. S4. Human Sdc1 expression constructs containing an intact ectodomain rescue spreading in mouse Sdc1 siRNA-transfected cells. FACS analysis for expression of (A) mouse Sdc1 (mAb 281.2) and (B,C) human Sdc1 mutants (mAb B-B4) – pLeuTM and Δcyto – against isotype IgG controls (red fill) 48 hours after transfection with either 600 nM control (Control) or mouse Sdc1-specific siRNA (siRNA). (D) B82L cells expressing human Sdc1 mutants were transfected with control or mouse Sdc1-specific siRNA and seeded on wells coated with either 5 μg/ml VN alone or a mixed substratum of VN plus 60 mg/ml of mAb B-B4. Cells were incubated at 37°C for 2 hours, fixed, and stained with Rhodamine-conjugated phalloidin. Bar, 50 μm.