Figure 9: Par3-exocyst interaction is necessary for cell survival.

(a) Interaction of SEC8 with wild-type YFP-PAR3, YFP-PAR3Δ(990-1018) or YFP-PAR3Δ(1014-1043). YFP-PAR3 constructs were transiently expressed in HEK293T cells and immunoprecipitated using an anti-Sec8 antibody, followed by immunoblotting with anti-GFP or anti-Sec8. (b) Confocal images showing localization of wild-type YFP-PAR3, YFP-PAR3Δ990-1018 or YFP-PAR3Δ1014-1043 in NMuMG cells; scale bars, 20 μm. (c) Western blots showing expression levels of YFP-Par3, YFP-PAR3Δ990-1018 or YFP-PAR3Δ1014-1043 expressed stably in NMuMG cells. β-catenin was used as loading control. Quantifications from three independent experiments and shown as average band intensity. Error bars, mean±s.e.m. (d) Representative DIC images of NMuMG cells expressing shLuc, shPar3 or shPar3 with wild-type YFP-PAR3b, YFP-PAR3bΔ(990-1018) or YFP-PAR3Δ(1014-1043) from three independent experiments. Scale bars, 50 μm. (e) Quantification of the percentage of cells dying; the graph shows mean±s.d. n=3. (f) Immunoblot showing pAkt and cleaved caspase-3 levels in NMuMG cells expressing shLuc, shPar3 or shPar3 with wild-type YFP-PAR3b, YFP-PAR3bΔ(990-10180 or YFP-PAR3Δ(1014-1043). (g) Quantification of three experiments shown in f. Error bars, mean±s.e.m. (h) Confocal images showing TJs in NMuMG and Eph4 cells. Wild-type cells or cells expressing YFP-PAR3bΔ(990-1018), or YFP-PAR3Δ(1014-1043) were transduced with shLuc or shPar3 lentivirus followed by paraformaldehyde fixation and staining with an anti-Occludin antibody. Scale bars, 20 μm. Image colours were inverted for a clearer depiction of Occludin structures. (i) E-cadherin localization in NMuMG cells expressing shPar3 with wild-type YFP-PAR3b, YFP-PAR3bΔ(990-1018) or YFP-PAR3Δ(1014-1043). Scale bars, 20 μm. The white dashed box represents the magnified region in the images. (j) Quantification of the PM/cytoplasmic ratio of E-cadherin; graph shows mean±s.d. Dotted line represents the average for shLuc expressing NMuMG cells. All experiments successfully repeated three times. P values were computed using one-way ANOVA followed by Dunnett’s multiple comparison tests.