Nef stimulates proliferation of glomerular podocytes through activation of Src-dependent Stat3 and MAPK1,2 pathways
J. Clin. Invest. John Cijiang He, et al. 114:643 doi:10.1172/JCI21004 [Go to this article.]

Figure 5
Role of Stat3 and MAPK1,2 in Nef-induced phenotypic changes in podocytes. Control vector–infected (V) or Nef-infected (Nef) podocytes were transfected with Stat3-DN. (A) Phospo-Stat3 and total Stat3 were determined by Western blot, with the following densitometric data: phospho-Stat3/total Stat3 ratio: V = 0.45 ± 0.15, Nef = 1.25 ± 0.20, V + Stat3-DN = 0.10 ± 0.05, Nef + Stat3-DN = 0.19 ± 0.06, P < 0.01 when Nef is compared with Nef + Stat3-DN, n = 3. (B) Cells were treated with PD98059 at 20 μM overnight, and MAPK1,2 activation was determined by Western blot, with the following densitometric data: phospho-MAPK1,2/total MAPK1,2 ratio: V = 0.91 ± 0.2, V + PD = 0.23 ± 0.11, Nef = 1.52 ± 0.32, Nef + PD = 0.34 ± 0.15, P < 0.01 when Nef is compared with Nef + PD, n = 3. (C) Cells were also used for determination of synaptopodin and GAPDH by Northern blot analysis and of cyclin E and β-actin by Western blot analysis, with the following densitometric data: synaptopodin/GAPDH ratio: V = 0.64 ± 0.21, V + PD = 0.70 ± 0.17, Nef = 0.17 ± 0.05, Nef + PD = 0.42 ± 0.12, Nef + Stat3-DN = 0.31 ± 0.05, Nef + PD + Stat3-DN = 0.63 ± 0.15, P < 0.05 when Nef is compared with Nef + PD or with Nef + Stat3-DN, P < 0.01 when Nef is compared with Nef + PD + Stat3-DN, n = 3; cyclin E/β-actin ratio: V = 0.28 ± 0.05, V + PD = 0.27 ± 0.1, Nef = 2.2 ± 0.52, Nef + PD = 1.8 ± 0.54, Nef + Stat3-DN = 1.32 ± 0.13, Nef + PD + Stat3-DN = 0.80 ± 0.22, P < 0.05 when Nef is compared with Nef + Stat3-DN, P < 0.01 when Nef is compared with Nef + PD + Stat3-DN, n = 4. (D) Cell proliferation: cells were transfected with Stat3-DN or treated with PD98059 as described above. Cell number was determined as described. n = 6. *P < 0.001 as compared with Nef-infected cells.