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Nanomolar potency and selectivity of a Ca2+ release-activated Ca2+ channel inhibitor against store-operated Ca2+ entry and migration of vascular smooth muscle cells
Article first published online: 22 AUG 2011
© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society
British Journal of Pharmacology
Volume 164, Issue 2, pages 382–393, September 2011
How to Cite
Li, J., McKeown, L., Ojelabi, O., Stacey, M., Foster, R., O'Regan, D., Porter, K. E. and Beech, D. J. (2011), Nanomolar potency and selectivity of a Ca2+ release-activated Ca2+ channel inhibitor against store-operated Ca2+ entry and migration of vascular smooth muscle cells. British Journal of Pharmacology, 164: 382–393. doi: 10.1111/j.1476-5381.2011.01368.x
- Issue published online: 22 AUG 2011
- Article first published online: 22 AUG 2011
- Accepted manuscript online: 24 MAR 2011 03:30AM EST
- Received; 4 October 2010; Revised; 16 February 2011; Accepted; 28 February 2011
Figure S1 Relevant gene expressions in human saphenous vein VSMCs. (a) Typical gel showing PCR products for Orai1, 2 and 3 mRNAs with (+) but not without (–) reverse transcriptase (RT) reaction. A DNA marker ladder is on the left. (b) Using real-time RT-PCR quantification, relative abundances of the mRNAs encoding the indicated proteins (n = 3–4).
Figure S2 Example Ca2+ add-back signals in human saphenous vein VSMCs with and without store depletion and showing comparison of effects of two chemically different store depletion agents, thapsigargin (TG) and cyclopiazonic acid (CPA). Intracellular Ca2+ was measured in cells pretreated (as indicated below) in Ca2+-free bath solution for 30 min and then exposed to 0.2 mM extracellular Ca2+ as indicated by the horizontal bar labelled ‘add-back’, except for one group of cells where the Ca2+ was not added back (0 Ca2+). The pretreatments in addition to the Ca2+-free solution were 1 μM TG, 10 μM CPA, 1 μM TG plus 10 μM CPA (TG + CPA), 0.2% dimethylsulphoxide (DMSO) as the vehicle control for TG and CPA, or no TG, CPA or DMSO (none).
Figure S3 Validation and specificity of siRNA knock down of Orai expression. See Table S1 for annotations. (a) Validation of knock down, showing relative abundances of Orai1, 2 and 3 mRNAs after transfection of vascular smooth muscle cells with Orai1, 2 and 3 siRNAs as indicated (n/N = 3/6). The control was scrambled siRNA. (b) Validation of specificity, showing lack of effect on the non-target mRNAs (as indicated) after transfection of vascular smooth muscle cells with the indicated siRNAs (n/N = 2–3/4–6).
Figure S4 Endogenous Orai1 protein detection and knock down in human saphenous vein VSMCs. Western blot for lysates from cells transfected with control (scrambled) siRNA (siRNA ctrl) or Orai1 siRNA 1 (Orai1 si.1). The blot was probed with anti-Orai1 antibody (upper panel) and anti-β-actin antibody (lower panel). The predicted protein mass of Orai1 is 33 kDa (arrow).
Figure S5 Typical original traces for the mean data shown in Figure 1A. For N = 16 per individual experiment, intracellular Ca2+ measurements from VSMCs pretreated with 1 μM thapsigargin in Ca2+-free bath solution for 30 min and then exposed to 0.2 mM extracellular Ca2+ as indicated by the horizontal bars: comparing control (scrambled) siRNA with Orai1 siRNA 1 (a), Orai2 siRNA (b), Orai3 siRNA (c), Orai2 and Orai3 siRNAs (d) and Orai1 siRNA 2 (e); and comparing DNA vector with Orai1 R91W mutant (dominant negative) (f).
Figure S6 In support of the dotted curve in Figure 2B, the graph shows the mean rate of rise data points without normalization and with the fitted Hill equation. The y-axis is the linear slope of the initial rate of rise of the fura-2 fluorescence after extracellular Ca2+ was added to the medium.
Figure S7 Original data for VSMC migration assays. Typical bright-field images of human saphenous vein VSMCs that had moved through pores in the polycarbonate membrane for vehicle control (ctrl) and 1 μM S66. A cell is indicated by a white arrow and a pore by a black arrow.
Table SI PCR primer pairs (F, forward direction; R, reverse direction), PCR amplicon sizes, and siRNA probes sequences. The ‘Orai1 si.negative’ was generated for the purpose of suppressing Orai1 expression, but it failed to have any effect on the expression and so was used as a negative control
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