Fragment N does not affect cell cycling.

<p>HeLa cells, infected with an empty virus or with a lentivirus encoding the HA-tagged version of the D157A fragment N mutant, were synchronized at G1 by treatment with mimosine for 18 hours. The cells were then washed and cultured in fresh medium for the indicated periods of time. <b>A.</b> Representative histograms obtained at different time points after release from the mimosine block (NT, not synchronized cells). <b>B.</b> Immunocytochemistry-based detection (gold staining) of fragment N in the infected cells. The nuclei are colored in blue (Hoechst staining). <b>C.</b> Percentage of cells in each phase of the cell cycle as determined by PI staining (DNA content). Results represent the mean ±95% CI of 4 independent experiments.</p

Fragment N does not regulate survivin expression.

<p><b>A.</b> MIN6 cells were co-transfected with a luciferase expressing vector under the control of either a minimal promoter sequence allowing the transcription of survivin (left graph) or the entire survivin promoter sequence (right graph) with increasing amounts of fragment N- (closed circles) or E2F1- (open circles) encoding plasmids. The results correspond to the mean ±95% CI of 6 (left panel) and 3 (right panel) independent experiments performed in triplicate. Repeated measures ANOVA tests were performed to determine if there was a significant increase in luciferase activity induced by the E2F1- or fragment N-encoding plasmids (normality was verified with the Shapiro-Wilk test). <b>B–C</b>. MEFs were infected with an empty virus or with a lentivirus encoding the HA-tagged version of the D157A fragment N mutant. Survivin mRNA levels were analyzed 24 hours later by quantitative RT-PCR (panel B). The location of the 672FW and 672RV primers (red arrows), used for the amplification of the survivin mRNA, is depicted on top of the graph. Alternatively, cells were lysed and the protein expression of HA-fragment N and survivin was assessed by Western blotting (panel C). The results correspond to the mean ±95% CI of 3 independent experiments. <b>D.</b> Skins of mice were irradiated with low (0.05 J/cm²) and high (0.3 J/cm²) doses of UV-B light. Expression of nuclear and cytoplasmic survivin was assessed by immunofluorescence <i>in situ</i> (left panel). The quantitation shown on the right-hand side corresponds to percentages of keratinocytes displaying nuclear or cytoplasmic survivin (mean ±95% CI of 6 and 10 mice for the low and high UV-B dose exposure, respectively). No cells were found to display both cytoplasmic and nuclear survivin expression.</p

Expression levels and functionality of the RIPN transgene in NOD mice.

<p><b>A.</b> Western blot analysis of lysates from islets isolated from ten week-old female mice. The presence of fragment N was assessed using an anti-HA antibody. <b>B.</b> Immunohistochemistry analysis of paraffin sections of five week-old female mice. Sections were stained using anti-insulin and anti-HA antibodies. Nuclei were stained with Hoechst 33342. <b>C.</b> Freshly isolated islets from 5 week-old females were incubated or not with inflammatory cytokines (1,000 units/ml TNFα, 1,000 units/ml interleukin-1β, and 50 units/ml interferon-γ) during 24 hours. The islets were then stained with Hoechst 33342 and apoptosis was scored.</p

Lymphocytic infiltration.

<p><b>A.</b> Paraffin sections of female mice of the indicated ages were stained with Hoechst 33342. Infiltration was scored as described in the methods. The upper images depict representative examples of the different infiltration grades (the white dotted lines delineate the islets and the red dotted lines encircle the lymphocytic infiltration). <b>B.</b> Paraffin sections of infiltration grade 2 islets of 10 weeks old NOD and NOD-RIPN mice were stained with an anti-CD3 specific antibody (orange). The nuclei were stained with Hoechst 33342 (blue). Quantitation of CD3-positive cells relative to the total number of islet cells is shown on the right. Note that T cells (i.e. CD3-positive cells) are smaller than islet cells. Hence T cells making about 50% of the total number of cells within islets occupy an islet area that is less than half.</p

Pancreatic histological sections from mice of the indicated age stained with anti-insulin antibodies and with Hoechst 33342 were used to determine the indicated parameters (see material and methods).

<p>Six female mice per genotype and per age were used (one section per animal). The results are represented as mean±95% CI. There was no statistical significant difference between NOD and NOD-RIPN mice as assessed by Student t test for any of the tested parameter.</p

Apoptosis <i>in situ</i>.

<p>Mice at the indicated ages were killed and apoptosis of insulin-expressing cells on islet sections was determined by the TUNEL assay. <b>A.</b> Examples of early and late apoptotic cells as well as apoptotic bodies (white arrowheads). The structure indicated by the red arrowhead is not considered as an apoptotic cell because it does not contain DNA (i.e. not stained with the Hoechst dye). Such structures are not included in the quantitation shown in panel B. <b>B.</b> Representative images of whole islet sections from 16 week-old animals stained for insulin and DNA and subjected to TUNEL. <b>C.</b> Quantitation of TUNEL-positive cells within the insulin-containing cell population. Results are derived from 5–6 mice per age group and genotype.</p

Diabetes development.

<p>The glycaemia of non-fasted mice (20 NOD females, 14 NOD-RIPN females, 16 NOD males, and 30 NOD-RIPN males) was measured once a week. Mice with glycaemia under 10 mM were considered normo-glycaemic and those with a glycaemia over 20 mM were considered diabetic. <b>A.</b> Normo-glycaemia curves. <b>B.</b> Diabetes-free curves. <b>C.</b> Glycaemia of individual mouse preceding the development of overt diabetes. Time 0 corresponds to the time when the mice had a glycaemia over 10 mM for the first time. <b>D.</b> Panel C was used to determine the percentage of mice that had glycaemia over 10 mM for the indicated number of weeks before becoming overtly diabetic.</p

Signals modulated by fragment N.

<p><b>A.</b> The extent of Akt activation was assessed by Western blot analysis of 10 µg of lysates from islets isolated from 5 week-old female mice using an antibody recognizing the phosphorylated form of Akt. Total levels of Akt were also determined using a non-phospho-specific anti-Akt antibody. <b>B.</b> Islets isolated from NOD and NOD-RIPN mice were stimulated or not for 30 minutes with inflammatory cytokines (1,000 units/ml tumor necrosis factor-α, 1,000 units/ml interleukin-1β, and 50 units/ml interferon-γ). The ability of nuclear proteins to interact with an NFκB-binding element-bearing radioactive probe was then monitored by EMSA as described in the methods. The locations of p65-p50 and p50-p50 complexes are indicated. The asterisk denotes a nonspecific band. <b>C.</b> INS1 cells were transfected with an empty vector (pcDNA3), a plasmid encoding the myristoylated active form of Akt (myr-Akt), a plasmid encoding a super NFκB repressor (IkBαΔN2), or a plasmid encoding fragment N in the indicated combinations, together with a NFκB-reporter luciferase construct and a GFP-encoding plasmid (to label the transfected cells). One day after, transfection, the cells were lysed to assess NFκB activity (left panel). Alternatively, apoptosis was scored in the transfected cells (right panel).</p