Figure 7.

PKC/Akt/NF-κB signaling pathway regulates flow-induced survivin expression and cell division. (a) After wild-type and cilia mutant (Pkd1^−/− and Tg737^Orpk/Orpk) cells were treated with PKC inhibitor or activator, both Akt and p-Akt were analyzed. When treated with PKC inhibitor, all cell lines showed down-regulation of p-Akt, while PKC activator treatment showed an increase in p-Akt compared to non-treated control cells. (b) The effect of fluid-flow on Akt and aurora-A expression was analyzed in the presence or absence of PKC inhibitor. When subjected to fluid-shear, p-Akt expression was up-regulated only in wild-type cells. While p-Akt expression returned to basal levels following treatment with PKC inhibitor and fluid-shear stress in wild-type cells, it stayed repressed in mutant cells. (c) Treatment with aurora-A inhibitor resulted in a decrease in p-Akt, aurora-A and survivin expression; however, these decreases were not significant from the control, non-treated group. (d) While total Akt level was not changed, fluid-shear stress significantly induced expression of p-Akt in wild-type but not in mutant cells. Aurora-A expression was increased following fluid-shear stress in wild-type cells; however, this increase was not significant from control. Both NF-κB and pNF-κB expressions were increased following fluid-shear stress only in wild-type cells, while mutant cells maintained a high basal level of NF-κB compared to static wild-type cells. Survivin expression was increased following shear-stress in wild-type cells. (e) Western blot analyses were conducted to confirm the signaling mechanism involving survivin expression by siRNA-mediated knockdown of PKC, Akt, aurora A, or survivin. To further confirm the involvement of these signaling molecules in centrosome number and cell division abnormality, immunofluorescence and flow cytometry analyses were presented in the Supplementary Materials together with the statistics.