FIGURE 6.

PKD family inhibitors prevent transient YAP translocation and phosphorylation in response to ANG II in IEC-18 cells. A, confluent cultures of IEC-18 cells were incubated in the absence (0) or presence of 5 μm Gö6983, 3.5 μm kb NB 142-70 (kb), or 2.5 μm CRT0066101 (CRT) for 1 h prior to stimulation of the cells without (−) or with 10 nm ANG II for 30 min. The cultures were then washed, fixed with 4% paraformaldehyde, and stained with an antibody that detects total YAP and with Hoechst 33342 to visualize the cell nuclei. B, quantification of the nuclear/cytoplasmic ratio of YAP immunofluorescence was determined using CellProfiler. The bars shown represent control (open bars), ANG II (red bars), ANG II + CRT (blue bars) and are the nuclear/cytoplasmic ratios from ∼1,000 cells from one experiment. Similar results were obtained in six independent experiments. C, confluent cultures of IEC-18 cells were incubated in the absence (0) or presence of 5 μm Gö6983 (Go), 3.5 μm kb NB 142-70 (kb), or 2.5 μm CRT0066101 (CRT) for 1 h prior to stimulation of the cells without (−) or with 10 nm ANG II for 30 min. Cells were then lysed with 2× SDS-PAGE sample buffer and analyzed by immunoblotting antibodies that detect YAP phosphorylated at Ser¹²⁷ and Ser³⁹⁷, PKD1 phosphorylated at Ser⁹¹⁶, PKDs phosphorylated at Ser⁷⁴⁴, and total PKD1/2 (PKD C-20), PKD3, YAP, and GAPDH. Similar results were obtained in six independent experiments. D and E, confluent cultures of IEC-18 cells were incubated in the absence (0) or presence of increasing concentrations of either CRT0066101 (closed squares, in E) or kb NB 142-70 (open squares in E) for 1 h prior to stimulation of the cells without (−) or with 10 nm ANG II for 30 min. Cells were then lysed with 2× SDS-PAGE sample buffer and analyzed by immunoblotting antibodies that detect YAP phosphorylated at Ser¹²⁷, total YAP, and GAPDH. Quantification YAP Ser¹²⁷ phosphorylation (shown in E) was performed using MultiGauge version 3.0. The results represent the mean ± S.E., n = 3, and are expressed as percentage of the maximum level of YAP Ser¹²⁷. E, confluent cultures of IEC-18 cells incubated in the absence (gray circle) or presence of increasing concentrations of CRT0066101 (closed circles) or kb NB 142-70 (open circles) for 1 h as indicated prior to stimulation of the cells without or with 10 nm ANG II for 30 min. The cultures were then washed, fixed with 4% paraformaldehyde, and stained with an antibody that detects total YAP and with Hoechst 33342 to visualize the cell nuclei and analyzed with the CellProfiler software. The results shown are the nuclear/cytoplasmic ratio of YAP immunofluorescence ± S.E.; ∼700 cells were analyzed at each concentration of CRT0066101 (closed circles) or kb NB 142-70 (open circles). Similar results were obtained in two independent experiments.