JCS02783 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1(Adobe PDF)
• Supplemental Table 2(Adobe PDF)
• Supplemental Figure 1 -

  Fig. S1. The amino-acid sequences (A) and phylogenetic tree (B) of the calcitonin family peptides, aligned for comparison. CLUSTAL W (1.82) multiple sequence alignment and phylogram of calcitonin, CGRP-a and -b, amylin, adrenomedullin and intermedin. Sequences of the fully processed peptides are boxed.

• Supplemental Figure 2 -

  Fig. S2. Glycosylation state of the transiently expressed CL receptors. (A) HEK293T cells were transfected with hCLpcDNA alone (lanes 1-3) or in the presence of RAMP1 (lanes 4-6) or RAMP2 (lanes 7-9) vectors. Cell lysates were treated with endoglycosidase F (lanes 2,5,8), endoglycosidase H (lanes 3,6,9) or vehicle  (lanes 1,4,7). Products were analysed by SDS-PAGE under non-reducing and reducing conditions and immunoblots were probed using LN-1436 antibody. The immunoblot is representative of three independent experiments. Arrowheads, deglycosylated (Mr ~37 kDa); open diamonds, core-glycosylated (~45 kDa); black diamonds, mature fully glycosylated (~55 kDa) forms of the receptor. The ~55 kDa hCL species, produced after co-expression with RAMPs, are reduced to a ~37 kDa hCL band after endoglycosidase F treatment, but were resistant to endoglycosidase H. For loading controls, the membrane was reprobed with anti-b-actin antibody.

• Supplemental Figure 3A
• Supplemental Figure 3B
• Supplemental Figure 3C
• Supplemental Figure 3D -

  Fig. S3. Localisation of endogenous CL in human tissues. (A-D) The localisation of hCL was assessed in human tissues by immunohistochemistry on paraffin sections from multiple tissue array. Rabbit polyclonal anti-hCL antibody LN-1436 (left image) and control pre-immune serum (right image) were used. Appropriate goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase were used (and further detected with DAB; brown colour; see Materials and Methods). Cell nuclei were counterstained with haematoxylin. Tissue and cell types expressing hCL were analysed by semi-quantification method (see supplementary material, Table S1). CL is expressed in microvascular endothelium (arrows) in the majority of the organs studied.

• Supplemental Figure 4 -

  Fig. S4. Localisation of endogenous CL in human tissues. (A-D) Double immunofluorescence was performed on frozen sections of human endometrium using combination of polyclonal anti-hCL antibody LN-1436 and monoclonal antibodies against specific markers of (A) endothelial cells (CD31), (B) smooth muscle cells (smooth muscle actin; SMA) and (C) leukocytes (CD45). (D) Control is pre-immune serum. The appropriate FITC- (for detection of hCL; left, first image) or Texas Red- (for detection of cell types within tissue; second image) conjugated secondary antibodies were used. DAPI (third image) was used to counterstain cell nuclei. Colocalized structures (right, fourth image) appear in yellow as determined by overlay of images.

• Supplemental Figure 5 -

  Fig. S5. Glycosylation state of the endogenous hCL receptors in vitro and in vivo. Microvascular endothelial and non-endothelial cell lines were grown as described in the Materials and Methods. (A) Total cell and (C) tissue lysates were analysed by SDS-PAGE under non-reducing and reducing conditions, and immunoblots were probed using antibody LN-1436. Arrowheads, deglycosylated (~37 kDa); open diamonds, core-glycosylated (~45 kDa); black diamonds, mature fully glycosylated (~55 kDa) forms of the receptor. (A) hCL is expressed specifically in primary MVECs. The origin of primary endothelial cells was confirmed by immunoblotting with anti-CD31 antibody. For loading controls, the membranes were reprobed with anti-b-actin antibody. (B) The ratio hCL/b-actin in cell lysates was calculated based on a densitometry values for the immunoblot bands. Endothelial cell lines: 1, human dermal MVECs; 3, human endometrial MVECs; 5, human myometrial MVECs; 7, human lung MVECs. Non-endothelial cell lines: 2, HEK293T fibroblast cell line; 4, MDA 231 human breast carcinoma epithelial cell line; 6, human normal myometrial smooth muscle cells; 8, human aortic vascular smooth muscle cells.

• Supplemental Figure 6 -

  Fig. S6. CL and RAMPs mRNA expression in primary human MVECs. Expression of CL and RAMPs mRNAs in primary endothelial cells was analysed by RT-PCR. RNA samples were from MVECs isolated from (1) skin, (2) lung, (3) myometrium and (4) endometrium. The set of primers for detection of b-actin (BA) was used for loading controls. (5) RNA sample from kidney served as a positive control.

• Supplemental Figure 7 -

  Fig. S7. Subcellular localisation of endogenous CL receptor in hDMVECs. Intracellular distribution of EndoCL was assessed by immunofluorescence using LN-1436 antibody and markers of individual cellular structures and organelles: (A) plasma membrane; (B) Golgi, (C) endoplasmic reticulum, (D) early sorting and (E) late endosomes, and (F) lysosomes (see Materials and Methods). (G) Control is rabbit pre-immune serum and mouse IgG. The appropriate FITC- (for the detection of hCL; left, first image) or Texas Red- (for the detection of subcellular structures and organelles; second image) conjugated secondary antibody were used. DAPI (third image) was used to counterstain cell nuclei. Colocalized structures (antigens; right, fourth image) appear in yellow as determined by overlay of images. Non-colocalised structures appear in green (green arrows) and red (red arrows). Figures are representative of three independent experiments.

• Supplemental Figure 8 -

  Fig. S8. Functional endogenous AM and CGRP receptors in microvascular endothelium. Cyclic AMP responses to the agonist stimulation in hDMVECs. Cells were treated with various concentrations of AM, CGRP, amylin (AMY) or VEGF. Forskolin (300 mM) was used as a positive control. Each point represents the mean ± s.e.m. of three experiments. (*P<0.05; **P<0.01; ***P<0.001).

• Supplemental Figure 9 -

  Fig. S9. Functional endogenous AM and CGRP receptors in microvascular endothelium. hDMVECs were seeded into the upper chamber of Transwell BD Biosciences inserts. Test peptide (human AM, CGRP and amylin) was added to the lower chamber. After 24 hours incubation, the cells were labelled with a fluorescent dye (calcein^-AM). Only those labelled cells that have migrated through the pores of the FluoroBlok membrane can be detected by inverted microscopy. The number of cells migrated to the lower surface of the insert was determined by measurement of fluorescence of invaded cells in a fluorescence plate reader with bottom reading capabilities. Full microvascular endothelial growth medium and VEGF were used as positive controls. Each point represents the mean ± s.e.m. of five separate experiments. (*P<0.05; **P<0.01; ***P<0.001).

• Supplemental Figure 10 -

  Fig. S10. (A) FACs analysis of MVECs using polyclonal antibody LN-1436. All sorted cells express Endo-hCL (green) as compared with pre-immune serum control (grey). (B) Subcellular distribution of Endo-hCL in MVECs before and after exposure to 100 nmol/l ligand (+AM or +CGRP) for 30 minutes. Subcellular distribution of the receptor was assessed by immunofluorescence microscopy using rabbit anti-hCL antibody and appropriate markers for individual cellular organelles (see Materials and Methods). Colocalisation was determined by overlay of the images on Openlab software. Cells were imaged with a Leica DMRBE microscope (Leica). Percentage of cells containing cell-surface-expressed (left bar) or internalised (right bar) receptor was quantified. MVEC-expressed Endo-hCL is internalised in response to adrenomedullin but not CGRP.