JCS025817 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Table S1. FACS analysis of MT1-MMP presence at cell surface.HUVECs that had been treated for 65 hours with siRNA were detached and stained with MT1-MMP Ab and Alex-Flour-488-conjugated anti-mouse IgG. Fluorescent samples were analysed in a flow cytometer. The mean fluorescence intensity relative to the control (control siRNA-transfected cells) is increased in p190A-KD cells and decreased in p190B-KD cells. As positive control, we used MT1-MMP−KD cells.

• Supplemental Figure S1a-c
• Supplemental Figure S1d -

  Fig. S1. Silencing p190A and B did not alter cell morphology. (A) HUVECs were transfected with the indicated siRNA targeting either p190A (A1), p190B (B1) or both (A1+B1). After 65 hours, cells were photographed. (B) Area and perimeter of siRNA-transfected cells were measured using Lucia software (Nikon). No major alteration was observed in p190-KD cells when compared with control. (C) Knockdown of p190 isoforms did not alter the actin cytoskeleton. HUVECs were transfected with indicated siRNA, fixed and stained for F-actin. No major reorganisation of the actin cytoskeleton was observed in p190-KD cells. (D) Knockdown of p190 isoforms did not alter focal adhesions. HUVECs were transfected with indicated siRNA, fixed and stained for F-actin and vinculin. For better visualisation, the colours of the images were modified in Photoshop. No major reorganisation of focal adhesions was observed in p190-KD cells when compared with control cells. Scale bars: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. PMA induced podosome structures in HUVECs. HUVECs were treated with PMA for 1 hour, fixed and permeabilised. F-actin was labelled with Alexa-Fluor-633−phalloidin (red) and vinculin with anti-vinculin and secondary Alexa Fluor 568 antibodies (green). For better visualisation, the colours of the images were modified in Photoshop. High-magnification images show the presence of individual podosomes defined by the presence of vinculin rings (arrows). Scale bar: 10 µm.

• Supplemental Figure S3ab
• Supplemental Figure S3cd -

  Fig. S3. p190A and p190B localised to podosomes in porcine aortic endothelial and smooth muscle cells. F-actin was labelled using Rhodamine-phalloidin and p190 isoforms were labelled using anti-isoform-specific antibodies and secondary Alexa-Fluor-488 antibodies in PAEV12Cdc42 (A) not treated or (B) treated with IPTG and in A7r5 (D) or not treated (C) or (D) treated with PMA. p190A and B localised to ruffles (A,C) and podosomes (B,D). Scale bar: 10 µm.