Journal: Biochemical Journal

Article Title: Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis

doi: 10.1042/BJ20131422

Figure Lengend Snippet: ( A ) PathHunter™ eXpress growth-arrested EDG-1 CHO-K1 β-arrestin cells were stimulated with 10 nM S1P in the presence of the indicated concentrations of NOX-S93. The activity of the reporter enzyme, β-galactosidase, was measured and plotted against the Spiegelmer® concentration as a percentage activity with the largest value set to 100%. In this representative experiment NOX-S93 inhibited the S1PR1 mediated effects of S1P with an IC 50 of 10.3 nM (means±S.D. of triplicate assays). ( B ) Intracellular calcium signalling in stably transfected CHO cells expressing S1PR3 and G α15 was stimulated with 10 nM S1P in the presence of the indicated concentrations of NOX-S93. Increase in intracellular calcium was measured and plotted against the Spiegelmer® concentration as a percentage with the largest value set to 100%. NOX-S93 (●) inhibited the S1P-induced calcium increase with an IC 50 of 5.45±0.89 nM ( n =4). The graph is representative of the result of one experiment (means±S.D. of triplicate assays). The control Spiegelmer® revNOX-S93 (■) showed no inhibition ( n =3).

Article Snippet: D - and L -RNA ( L -amidites were from Chem-Genes) were synthesized at NOXXON by standard phosphoramidite chemistry and the final Spiegelmer® candidate was conjugated to a 40 kDa polyethylene glycol moiety (JenKem) via an aminohexyl linker at the 5′-end [ ] and named NOX-S93.

Techniques: Activity Assay, Concentration Assay, Stable Transfection, Transfection, Expressing, Control, Inhibition

Journal: Biochemical Journal

Article Title: Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis

doi: 10.1042/BJ20131422

Figure Lengend Snippet: ( A ) HUVEC spheroids were embedded in a 3D collagen gel, stimulated with 100 nM S1P and incubated for 24 h with different concentrations of NOX-S93. The mean±S.D. of the cumulative sprout length of ten randomly selected spheroids per data point is depicted. −, basal sprouting without S1P stimulation and NOX-S93 treatment as a negative control. ( B ) IC 50 of NOX-S93 for the inhibition of HUVEC spheroid sprouting after stimulation with 100 nM S1P. ( C ) NOX-S93 IC 50 values for the inhibition of HUVEC spheroid sprouting after stimulation by different growth factors. The control Spiegelmer® revNOX-S93 showed no inhibition.

Article Snippet: D - and L -RNA ( L -amidites were from Chem-Genes) were synthesized at NOXXON by standard phosphoramidite chemistry and the final Spiegelmer® candidate was conjugated to a 40 kDa polyethylene glycol moiety (JenKem) via an aminohexyl linker at the 5′-end [ ] and named NOX-S93.

Techniques: Incubation, Negative Control, Inhibition, Control

Journal: Biochemical Journal

Article Title: Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis

doi: 10.1042/BJ20131422

Figure Lengend Snippet: HUVECs were cultured in 2D, serum-starved and stimulated with 100 nM S1P in the presence or absence of NOX-S93 or the non-functional control Spiegelmer® revNOX-S93 for 5 min. Activation of ERK1/2 was studied by Western blotting using a phosphospecific antibody. Whereas a 10-fold excess of NOX-S93 completely inhibited S1P-induced ERK1/2 phosphorylation, the control Spiegelmer® revNOX-S93 was inactive.

Article Snippet: D - and L -RNA ( L -amidites were from Chem-Genes) were synthesized at NOXXON by standard phosphoramidite chemistry and the final Spiegelmer® candidate was conjugated to a 40 kDa polyethylene glycol moiety (JenKem) via an aminohexyl linker at the 5′-end [ ] and named NOX-S93.

Techniques: Cell Culture, Functional Assay, Control, Activation Assay, Western Blot, Phospho-proteomics