<i>Fli1</i>^<i>+/-</i> T cells have significantly lower levels of Neuraminidase 1 (<i>Neu1</i>) message and NEU activity compared to <i>Fli1</i>^<i>+/+</i> T cells during early disease.

<p>cDNA was amplified from RNA isolated from T cells of MRL/lpr <i>Fli1</i>^<i>+/+</i> and <i>Fli1</i>^<i>+/-</i> 10-12 week-old mice (A) and 17-18 week-old mice (C). <i>Neu1</i> and <i>Neu3</i> message levels were measured by real-time PCR and normalized to <i>β-actin</i> levels. B) NEU activity was measured as described in the methods. Relative levels in the NEU activity assay were calculated to combine all animals across experiments as described in the methods. The ‘n’ represents data from individual animals and p values are provided within the figure.</p

<i>Fli1</i>^<i>+/-</i> T cells have significantly lower levels of Neuraminidase 1 (<i>Neu1</i>) message and NEU activity compared to <i>Fli1</i>^<i>+/+</i> T cells during early disease.

<p>cDNA was amplified from RNA isolated from T cells of MRL/lpr <i>Fli1</i>^<i>+/+</i> and <i>Fli1</i>^<i>+/-</i> 10-12 week-old mice (A) and 17-18 week-old mice (C). <i>Neu1</i> and <i>Neu3</i> message levels were measured by real-time PCR and normalized to <i>β-actin</i> levels. B) NEU activity was measured as described in the methods. Relative levels in the NEU activity assay were calculated to combine all animals across experiments as described in the methods. The ‘n’ represents data from individual animals and p values are provided within the figure.</p

Glycosphingolipid levels are significantly reduced in T cells from late disease stage MRL/lpr <i>Fli1</i>^<i>+/-</i> compared to MRL/lpr <i>Fli1</i>^<i>+/+</i> mice.

<p>Glycosphingolipids lactosylceramide (LacCer) (A) and glucosylceramide (GluCer) (B) were measured by SFC/MS/MS in unstimulated or anti-CD3/CD28 stimulated T cells isolated from 17–18 week-old MRL/lpr mice. *p<0.05, **p<0.01, #p<0.005, # #p<0.001. The ‘n’ represents data from individual animals.</p

Both the mouse and human <i>Neu1</i> promoters respond to FLI1 in a dose-dependent manner.

<p>Promoter/reporter constructs pGL3 <i>mNeu1</i> containing the mouse promoter (A) or pSG <i>hNEU1</i> containing the human promoter (B) were transfected with increasing amounts of a FLI1 expression vector into the mouse S1A (A) or human Jurkat (B) T cell lines. Transfection efficiency was adjusted for by expression from a co-transfected normalization plasmid. Results are representative of four independent transfections with similar results.</p

Intracellular Ca²⁺ levels are significantly reduced in <i>Fli1</i>^<i>+/-</i> T cells compared to <i>Fli1</i>^<i>+/+</i> T cells from early disease stage mice following TCR-specific activation.

<p>T cells from the spleens of MRL/lpr <i>Fli1</i>^<i>+/+</i> and <i>Fli1</i>^<i>+/-</i> 10-12 week-old (A and B) and 17-18 week-old (C and D) mice were stimulated with anti-CD3/CD28 (A and C) or PMA/ionomycin (B and D) and Ca²⁺ influx measured over time. PMA/ion was added 5 seconds after beginning data collection and data was collected for 1 min. Anti-CD3/CD28 antibodies were added just before beginning data collection and data was collected for 45 minutes. All data collection points are presented from 0–60 sec for PMA/ion and 0-45 minutes for anti-CD3/CD28. The ‘n’ represents data from individual animals and p values are provided within the figure.</p

FLI1 levels have no effect on cell death of MRL/lpr T cells following TCR-specific activation.

<p>T cells from the spleens of MRL/lpr <i>Fli1</i>^<i>+/+</i> and <i>Fli1</i>^<i>+/-</i> 10-12 week-old mice were stimulated with anti-CD3/CD28 for 0, 3 and 24 hours and cell death measured by flow cytometry following Annexin V and PI staining. A) Annexin V+/PI- (apoptosing cells), B) Annexin V+/PI+ (dead cells), and C) Annexin V-/PI- (live cells). The ‘n’ represents data from individual animals.</p