Quantitative Reverse Transcriptase Primers.

<p>Quantitative Reverse Transcriptase Primers.</p

Activation of the IL-2 receptor is associated with induction of JAK/STAT protein expression.

<p>Expression of JAK 3, pJAK 3, STAT 5a, and pSTAT 5 in non-stimulated podocytes were compared to podocytes stimulated with IL-2. (A)There is increased expression of JAK 3 after both 30 and 60 minutes of IL-2 stimulation (P<0.05, One-way ANOVA with Tukey’s multiple comparisons test). N = 3 in individual groups. (B) Phosphorylated JAK 3 is significantly increased after 30 minutes of stimulation with IL-2 (*, P<0.01); however, after 60 minutes the expression has returned to within unstimulated levels (**, P<0.01; One-way ANOVA with Tukey’s multiple comparisons test). N = 3 in individual groups. (C) Cytoplasmic STAT 5a protein expression is significantly increased in podocytes after 60 minutes of IL-2 stimulation (P<0.0001, One-way ANOVA with Tukey’s multiple comparisons test) compared to unstimulated podocytes. N = 3 in individual groups. (D) Nuclear STAT 5a protein expression in podocytes was significantly decreased at both time points with IL-2 stimulation (*, P<0.0001) compared to unstimulated podocytes. However, podocytes stimulated for 60 minutes showed a significantly higher STAT 5a protein expression when compared to 30 min (**, P<0.0001; One-way ANOVA with Tukey’s multiple comparisons test). N = 3 in individual groups. (E) Cytoplasmic phosphorylated STAT 5 protein expression was significantly decreased following 30 minutes of stimulation with IL-2, but this effect was not sustained after 60 minutes of stimulation (P<0.05, One-way ANOVA with Tukey’s multiple comparisons test). N = 3 in individual groups. (F) Similar to phosphorylated JAK3, phosphorylated nuclear STAT 5 protein expression was significantly increased after 30 minutes of IL-2 stimulation, but was significantly decreased after 60min of IL-2 stimulation compared to both unstimulated podocytes and podocytes stimulated with IL-2 for 30 minutes (Fig 4F). (P<0.001, One-way ANOVA with Tukey’s multiple comparisons test). N = 3 for individual groups. Bar columns and error bars represent mean and standard deviation respectively.</p

Antibody list for western blot.

<p>Antibody list for western blot.</p

IL-2 activates the podocyte mitochondrial intrinsic pathway and inhibits podocyte autophagy.

<p>The induction of apoptosis was mediated by the intrinsic pathway as reflected by the increased expression of (A) Bax (P<0.05, unpaired t-test) and (B) cFLIP (P<0.01, unpaired t-test). IL-2 resulted in inhibition of podocyte autophagy with decreased protein expression for LC3 II (P<0.05). N = 4 for individual groups. Bar columns and error bars represent mean and standard deviation respectively.</p

IL-2 increases podocyte paracellular albumin permeability.

<p>Murine podocytes (JR07) were seeded onto transwell filters (3μm pore, Corning) at (5–6 X 10³ cells/well) and allowed to differentiate for 10 days. Compared with the controls, IL-2 treatment resulted in a greater albumin influx across the podocyte monolayer (P<0.05, One-way ANOVA). N = 3 for individual groups. Bar columns and error bars represent mean and standard deviation respectively.</p

Stimulation with IL-2 increases mRNA expression of STAT 5 subunits.

<p>Expression of STAT 5a-b subunits in non-stimulated podocytes were compared to podocytes stimulated with IL-2. The mRNA expression of STAT 5a-b was significantly increased after stimulation with IL-2 (P< 0.01, P<0.05 respectively; One-way ANOVA). N = 3 in individual groups. Bar columns and error bars represent mean and standard deviation respectively.</p

IL-2 induces podocyte mitochondrial depolarization and apoptosis.

<p>Flow cytometry measured mitochondrial membrane potentials and apoptosis in podocytes. (A) IL-2 induces a significant increase in mitochondrial membrane depolarization. (B) Incubation with IL-2 also resulted in increased apoptosis (P< 0.05, unpaired t-test). N = 4 for individual groups. Bar columns and error bars represent mean and standard deviation respectively.</p

Stimulation with IL-2 increases mRNA expression of all three subunits of the IL-2R in differentiated murine podocytes.

<p>All the subunits were detected by qRT-PCR within 30 minutes after stimulation with IL-2. There are significant increases in the expression of the three subunits when normalized to GAPDH (P< 0.05). N = 3 in individual groups. P<0.05 (One-way ANOVA). Bar columns and error bars represent mean and standard deviation respectively.</p

IL-2R subunits are present in differentiated murine podocytes.

<p>(A) Representative flow cytometry histograms showing surface IL-2R subunits α and γ (black and green histograms). Nonspecific staining (black histogram) was determined using an isotype-specific control antibody. Murine podocytes express the three subunits of the IL-2R. (B) IL-2R α alpha and β chains are expressed in non-stimulated podocytes. Differentiated podocytes express α alpha and β mRNA subunits of the IL-2R as shown by real time PCR. (C) The alpha subunit is demonstrated by Western blot. The positive control consists of protein extracts obtained from Jurkat (JKT) cells. (D) The conditionally immortalized murine podocyte clone JR07 expresses podocyte specific proteins nephrin and podocin in proliferating and differentiating cells as demonstrated by Western blot. (E) Whole cell lysates of murine podocytes were immunoblotted with anti- alpha, beta, and gamma chains antibodies showing the presence of the whole complex of the IL-2R by Western blot. (F) IL-2R expression in podocytes from human renal biopsies. Immunohistochemical staining was performed on human renal biopsies from children with FSGS and control. CD25 (alpha chain) expression was observed in biopsies from children with FSGS on the surface podocytes, indicated by the arrows (left panel) when compared to the biopsy control (right panel).</p