Figure 7.
Claudin-2 silencing slows proliferation through GEF-H1/RhoA-dependent up-regulation of p27kip1. A–C, LLC-PK1 (A and B) or MDCK (C) cells were transfected with NR, Cldn-2, RhoA, or GEF-H1–specific siRNA, and the indicated proteins were detected and quantified by Western blotting. D, RhoA activation reduces proliferation. Confluent LLC-PK1 cells were treated with 0.25 mg/ml Rho activator II for 16 h, and BrdU incorporation was measured as in Fig. 6. Graphs show means ± S.D. In A, n = 10; in B, n = 4; and in D, n = 3. E, LLC-PK1 cells were transfected with NR or Cldn-2–specific siRNA with or without p27kip1 siRNA, as indicated, and BrdU incorporation was measured. In parallel experiments, silencing was verified by Western blotting (means ± S.D., n = 12). F, mice were sham-operated or underwent UUO (7 days), as described in Fig. 1. Renal tissue sections were processed for immunohistochemistry using a primary antibody against p27kip1. Quantification was done as described under “Experimental procedures” (n = 5 and 7 random areas in 2 animals). Bar, 50 μm.