JCS043174 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Intercellular lumens observed in PAR-3 kd cells cultured under normal growth conditions. Confluent PAR-3 kd stable clone (13-32) cells cultured on filter supports were fixed and stained for the indicated proteins. Arrows indicate intercellular lumens. (A) Projected views of confocal sections with enlarged z-sectional views. (B) A gallery of views of the confocal sections. Arrowheads indicate the position of the lateral membrane in each panel. Scale bars: 20 µm

• Supplemental Figure S2 -

  Fig. S2. Western blot data showing the specific reduction in PAR-3 expression in the PAR-3 kd cells used in this study. Total cell extracts of the indicated MDCK cells were subjected to western blot analysis using the antibodies shown on the right. PAR-3 kd #1 and #2 represent heterogeneous stable cells expressing PAR-3 shRNA sequences #1 and #2, respectively.

• Supplemental Figure S3 -

  Fig. S3. Western blot data showing equal expression levels of ectopically introduced wild-type PAR-3 (WT) and PAR-3 S827/829A. (A) Total cell extracts of PAR-3 kd clone (25a) cells infected with adenovirus expression vectors encoding the indicated proteins were subjected to western blot analysis using the antibodies shown on the right. (B) Total cell extracts of the cells used in Fig. 4D (PAR-3 kd cells +EGFP, +PAR-3 WT and +PAR-3 S827/829A) were subjected to western blot analysis using the antibodies shown on the right. For comparison, typical data for cells expressing sPAR-3 WT and sPAR-3 S827/829A are also presented (see Fig. 5).

• Supplemental Figure S4 -

  Fig. S4. MDCK cells infected with adenovirus vectors encoding the indicated proteins were cultured on filter supports and subjected to a CS. At 3 hours after the CS, the cells were immunostained for the indicated components to examine both apical domain re-establishment and junction reassembly. Projected views of confocal sections are presented. Note that overexpression of PAR-3 S827/829A does not exert any deleterious effects on apical domain development. Scale bars: 20 µm.

• Supplemental Figure S5 -

  Fig. S5. Western blot data showing the expression levels of ectopically introduced mLgl2, aPKCλ, aPKCλ KN, PAR-6β and PAR-6β M235W. Total cell extracts of MDCK cells stably expressing the indicated proteins were subjected to western blot analysis using the antibodies shown on the right.

• Supplemental Figure S6 -

  Fig. S6. Specificity of the anti-PAR-6 polyclonal antibody used in this study. (A) pEB-based RNAi stable transformants of MDCK cells were established using two independent shRNA sequences (#2 and #5) for canine PAR-6β. Total cell extracts were subjected to western blot analysis using the anti-PAR-6β antibody. (B) Total cell extracts of MDCK cells overexpressing T7-tagged PAR-6α, β or γ were analyzed with the anti-PAR-6β antibody (right). The amount of each sample loaded was adjusted to normalize the expression levels of each T7-tagged PAR-6 (left). To indicate the position of endogenous PAR-6β, a HeLa cell extract was loaded in lane 1. (C) MDCK cells were transiently cotransfected with two expression vectors encoding EGFP and an shRNA sequence for canine PAR-6β, respectively. Cells were cultured on filter supports for 3 days, and then stained with the anti-PAR-6β antibody. Note that the antibody stains the apical domain and apical junctional complex as reported previously (Yamanaka et al., 2003), and these signals disappear in EGFP-expressing cells.

• Supplemental Figure S7 -

  Fig. S7. Immunogold electron microscopy data showing the localization of aPKC on the cytoplasmic surface of VACs. (A) PAR-3 kd stable clone (13-32) cells were highly depolarized by prolonged incubation (20 hours) in low-calcium medium, and then subjected to immunogold microscopy using an anti-aPKC antibody. N, nucleus; V, VAC. (B) Enlarged view of the framed region in A. The arrows and bracket indicate the positions of gold particles. Scale bars: 0.5 µm.