Dual Interaction of JAM-C with JAM-B and _M2 Integrin: Function in Junctional Complexes and Leukocyte Adhesion

Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 1 - JAM-C is not internalized when engaged with JAM-B at inter-cellular contacts.

  CHO cells transfected with EGFPJAM-C cells and cocultured with JAM-B or JAM-C expressing cells were stained with a monoclonal antibody against JAM-C and anti-rat coupled to phycoerythrin. Negative controls and compensation settings were established with unstained or single stained cell populations. Negative control (black profile) and surface expression of EGFPJAM-C after coculture with JAM-C expressing cells (dashed profile) or JAM-B expressing cells (filled profile) are shown. No significant loss of surface staining is observed when EGFPJAM-C transfected cells were cultured with JAM-B as compared to JAM-C expressing cells. One representative experiment is shown.

• Supplemental Figure 2 - (A) Representative plots of apparent whole-cell weight-average molecular weight (Mw) against sample concentration (mg/ml) for absorbance data at 280 nm and 10 000 r.p.m. (B) Representative plots of apparent whole-cell weight-average molecular weight (Mw) against sample concentration (mg/ml) for absorbance data at 280 nm and 18 000 r.p.m. Open blue circles are for JAM-B, red circles are for JAM-C and green diamonds are for an equimolar JAM-B/JAM-C mixture. Fitted curves are for a linear regression of weight with concentration as appropriate for a dimerization process (Ikemizu et al, 2000).
• Supplemental Figure 3 - The JAM-B/JAM-C heterodimerization requires an intact membrane distal V domain and is stabilized by the membrane proximal C₂ domain.

  MDCK (JAM-B-EGFP) cell lysates were precipitated using recombinant soluble JAM-C consisting in the V and C₂ extra-cellular domains (2d), the membrane distal V domain (1d), the two extra-cellular domains mutated at position 66 (E66R 2d) or the membrane distal domain mutated at position 66 (E66R 1d). The soluble form of JAM-C E66R 2d shows a reduced binding to JAM-B, as compared to the intact soluble form 2d. In addition the mutated soluble V domain alone (E66R 1d) is not able to interact with JAM-B. These results indicate that the membrane distal V domain of JAM-C represents the minimal requirement to get interaction with JAM-B. Moreover, the C2 domain of JAM-C probably stabilizes this interaction.

• Supplemental Figure 4 - LyEND.1 cells are PymT-transformed endothelioma cells derived from lymphatic vascular tumors.

  (A, B) Newborn CD1 mice were injected intra-peritoneally with 5x10⁴ freshly prepared MSCV-PymT(245Δ10) retroviral particles. Inoculated mice were monitored daily for signs of hemorrhages or bleeding and sacrificed 20 days after inoculation. Within 20 days after injection the development of classical hemangiomas, which appear as blood filled cysts (A) was observed, as well as several tumors that were filled with a clear translucent fluid (B). Macroscopically visible tumors were excised, dissociated and cultured in DMEM containing 20 % FCS. Islets of cells with typical endothelial morphology were expanded to homogeneously growing cell lines in DMEM containing 10 % FCS. (C, D) The LyEnd.1 cells were analyzed by immunocytochemistry. LyEnd.1 cells express the endothelial marker proteins PECAM-1 (C) and VE-cadherin (D) at the cell surface. Nuclei were counterstained using Hoechst H33258. (E-G) Flow cytometry analysis of LyEnd.1 cells. In addition to PECAM-1 expression (E) we detected moderate levels of ICAM and low levels of VCAM expression by flow cytometry (F, G). Neither ICAM nor VCAM cell surface expression was up-regulated after TNF-α exposure of LyEnd.1 cells (green line, non-treated cells, red line, TNF-α-treated cells). (H) Given the presumably lymphatic origin of the LyEnd cell lines we probed for the presence of prototypic lymphatic markers in these cells using RT-PCR. We detected mRNAs coding for the cell surface molecules Lyve1 and Flt4 (H) as well as mRNAs for the nuclear transcription factor Prox1 (data not shown). Low abundance message for the cell surface protein Podoplanin/T1A was also detected. From these data we conclude that the endothelioma cell line LyEnd.1 expresses a number of markers proteins indicative of endothelial cells and in addition has retained expression of markers characteristic of lymphatic endothelium.