FIGURE 5.

ANG II induces cytoplasmic localization and phosphorylation of YAP through an Akt-independent but PKC-dependent pathway in IEC-18 cells. A, confluent cultures of IEC-18 cells were incubated in the absence (0) or presence of 2.5 μm GSK690693 (GSK), 2.5 μm GDC-0068 (GDC), or 5 μm MK2206 (MK) 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 shown in A was determined with the CellProfiler software. The bars shown are the mean nuclear/cytoplasmic ratio ± S.E. n = 6 fields (∼1,200 cells were analyzed for each condition). Similar results were obtained in three independent experiments. C, confluent cultures of IEC-18 cells were incubated in the absence (0) or presence of 2.5 μm GSK690693 (GSK), 2.5 μm GDC-0068 (GDC), or 5 μm MK2206 (MK) 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 with antibodies that detect YAP phosphorylated at either Ser¹²⁷ or Ser³⁹⁷, PRAS40 phosphorylated at Thr²⁴⁶, and total YAP and GADPH. Similar results were obtained in three independent experiments. D, confluent cultures of IEC-18 cells were incubated in the absence (0) or presence of 2.5 μm GSK690693 (GSK), 5 μm Gö6983 (Go), 5 μm GF1, or 10 μm H89 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. E, quantification of the nuclear/cytoplasmic ratio of YAP immunofluorescence shown in D was determined with the CellProfiler software. The bars shown are the mean nuclear/cytoplasmic ratios ± S.E. n = 6 fields (∼1,100 cells were analyzed for each condition). Similar results were obtained in four independent experiments. F, confluent cultures of IEC-18 cells were incubated in the absence (0) or presence of 2.5 μm GSK690693 (GSK), 5 μm Gö6983 (Go), 5 μm GF1, or 10 μm H89 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 and total YAP. Similar results were obtained in three independent experiments.