Figure 8.

Cldn-2 silencing activates MRTF and enhances pro-fibrotic changes in tubular cells. A and B, WT or HA–CLDN-2 expressing LLC-PK₁ cells were transfected with a firefly luciferase-coupled MRTF/SRF sensitive promoter (3DA), along with the internal control pRL-TK (Renilla luciferase). In A, the cells were also transfected with NR or Cldn-2–specific siRNA. B, cells were treated with the Rho inhibitor Rhosin (24 h, 30 μm). 24 h post-transfection luciferase activity was determined using the Dual-Luciferase assay kit. Firefly luciferase activity was normalized using the Renilla luciferase activity and expressed as fold change from control (means ± S.D., n = 3) C, whole cell extracts were separated on 6% SDS gels to detect the relative shift in MRTF-A molecular mass with α-actinin as loading control. D, LLC-PK₁ cells were transfected with Cldn-2 or MRTF-A–specific siRNAs, or both, as indicated. CTGF and SMA were detected and quantified (means ± S.D., n = 3). The blots were developed for Cldn-2 and MRTF-A to verify silencing. E and F, LLC-PK₁ cells were transfected with siRNAs and treated with 10 ng/ml TGFβ1 for 1 h (in E) or 24 h (E and F), and the indicated proteins were detected. Note that the two blots in E were run parallel, and thus the left blots serve as control for detectability of SMA expression. In F, graph shows means ± S.D., n = 3.