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P.J. Noy et al.
Silencer Select siRNA duplexes (Invitrogen) using Lipofectamine RNAiMAX (Invitrogen).
Quantitative real-time polymerase chain reaction
Quantitative real-time polymerase chain reaction (qPCR) was performed using TaqMan assays for Tspan18, Orai1, Orai2, Orai3, 18S and GAPDH.30 Details are available in the Online Supplementary Appendix.
Lentiviral transduction
Human umbilical vein endothelial cells were lentivirally trans- duced with Orai1-Myc as described.31 Details are available in the Online Supplementary Appendix.
brane structure.4 For example, no Tspan18 antibodies have been published, and commercially-available anti- bodies are made to Tspan18 peptides and do not detect full-length Tspan18 when rigorously tested (MG Tomlinson, 2019, unpublished manuscript). Therefore, to characterize the Tspan18 expression profile, mouse tis- sues were tested by qPCR. Tspan18 mRNA was most highly expressed in lung and at lower levels in other tis- sues (Figure 1A). Analyses of published transcriptomic data40 showed that Tspan18 was most highly expressed by endothelial cells compared to other mouse lung cell types (Figure 1B). Similar analyses of transcriptomic data from mouse brain41 also showed relatively strong endothelial expression of Tspan18 (Figure 1C). Consistent with this, qPCR showed that Tspan18 was expressed by primary HUVEC and the human microvas- cular endothelial HMEC-1 cell line (Figure 1D). Tspan18 expression was low or absent on most other cell types tested, although peripheral blood leukocytes expressed comparable levels to HUVEC (Figure 1D). In transcrip- tomic data from the Human Protein Atlas (www.proteinat- las.org), Tspan18 was expressed by most human tissues at a level between 10 and 70 tags per million, but in cell lines was only expressed at 10 or greater tags per million by HUVEC and 8 of the other 64 cell types analyzed.42
Tspan18 is required for Ca2+ signaling in primary human endothelial cells
To investigate Tspan18 function, its expression in HUVEC, which is a widely-used primary human endothelial cell model, was knocked-down using two dif- ferent siRNA duplexes. Subsequent analyses revealed a 60% reduction in peak Ca2+ elevation in response to the inflammatory mediator thrombin (Figure 2A). A similar defect was observed in response to histamine (Figure 2B). Positive control ionomycin treatment gave a sustained intracellular Ca2+ response in all samples (Figure 2C) and effective knockdown was confirmed by qPCR (Figure 2D). Functionality of thrombin and histamine receptors was confirmed by anti-phospho-ERK1/2 mitogen-activat- ed protein kinase (MAPK) western blotting, as this was not affected by Tspan18 knockdown (Figure 2E).
Tspan18 promotes Ca2+-responsive nuclear factor of activated T-cell signaling in lymphocyte cell lines
To investigate the mechanism by which Tspan18 regu- lates Ca2+ signaling, a more tractable cell line system was established, namely DT40 cells that are derived from chicken B cells. In this cell line, a transfected NFAT/adapter protein 1 (AP-1) transcriptional luciferase reporter can be used as a readout for Ca2+ signaling down- stream of transfected membrane proteins.21,43 Transfection of a FLAG epitope-tagged Tspan18 expres- sion construct was sufficient to induce robust NFAT/AP-1 activation (Figure 3A). As controls, five other FLAG- tagged tetraspanins (CD9, CD63, CD151, Tspan32 and Tspan9) were chosen because they represent a diverse range of tetraspanins based on sequence identities.22 These did not induce NFAT/AP-1 activation, despite their substantially higher expression than Tspan18 as assessed by anti-FLAG western blotting (Figure 3A).
Despite the fact that the NFAT/AP-1 promoter can be activated by Ca2+ signaling, it is maximally activated by combined Ca2+ signaling and MAPK; Ca2+ activates NFAT and MAPK activates AP-1. To determine whether
Nuclear factor of activated T-cell/AP-1-luciferase transcriptional reporter assay
The NFAT/AP-1-luciferase assay, and β-galactosidase assay to normalize for transfection efficiency, were as described.21
Co-immunoprecipitation
A detailed description of co-immunoprecipitation from trans- fected HEK-293T cell lysates22 is provided in the Online Supplementary Appendix.
Immunofluorescence microscopy
Detailed information is provided in the Online Supplementary Appendix. In brief, cells were prepared as described29 and the Manders’ coefficients (M1 and M2) were used as the co-localization measure.32 Ear vasculature was imaged and quan- tified as described.33,34
Immunohistochemistry
Immunohistochemistry was as described;35 details are avail- able in the Online Supplementary Appendix.
Intracellular Ca2+
Human umbilical vein endothelial cells were loaded with
Fluo-4 NW dye according to the manufacturer’s instructions (Molecular Probes). Fluorescence was measured every 3 seconds for 5 minutes using a FlexStation fluorescence reader (Molecular Devices), and thrombin (1 U/mL), histamine (20 μM) or iono- mycin (10 μM) were injected after acquiring a baseline for 30 seconds.
ELISA and coagulation time assays
Detailed information is provided in the Online Supplementary Appendix.
Platelet aggregation and adhesion to human umbilical vein endothelial cells
Platelet assays were as described;36,37 detailed information is provided in the Online Supplementary Appendix.
In vivo assays
Mouse models were as described;13,37-39 detailed information is
in the Online Supplementary Appendix. Results
Tspan18 is expressed by endothelial cells
A lack of effective antibodies to many tetraspanins is a current problem in the tetraspanin field. This may be due to their relatively small size, high degree of sequence con- servation during evolution, and compact 4-transmem-
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haematologica | 2019; 104(9)