JCS02938 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Calcium-switch-mediated localizations of ZO-1, E-cadherin and ezrin in mLgl kd cells are almost similar to those of control cells. mLgl kd cells (24-15) or control cells (21-7) were incubated with LC medium for 20 hours, after which the cells were subjected to calcium switch for 3 or 24 hours. The cells were then fixed and stained with antibodies for the indicated proteins. Notice that the localization of ZO-1 as well as E-cadherin or ezrin was almost the same as that of control cells. Photographs represent the projected views of confocal sections. Bars, 20 mm.

• Supplemental Figure 2 -

  Fig. S2. Effects of overexpression of mLgl or PAR-3 on redistribution of gp135 in response to depolarization of MDCK cells. (A, B) Control cells (21-1) or mLgl kd cells (24-15) on permeable filters were infected with adenovirus expression vectors encoding LacZ or HA-mLgl-2 for 2 hours. The cells were then incubated with LC medium for 18 hours. (A) Cells were fixed and double stained with anti-mLgl-2 and anti-gp135 antibodies. (B) Quantification of cells with cell peripheral gp135 staining in A. Notice that the overexpression of HA-mLgl-2 decreased the number of cells with peripheral gp135 stainings not only in control cells but also in mLgl kd cells. (C, D) Control cells (1-5) or PAR-3 kd cells (25a) cultured on permeable filters were infected with adenovirus expression vectors encoding LacZ or T7-PAR-3 for 2 hours. The cells were then incubated with LC medium for 18 hours. (C) Cells were fixed and double stained with anti-PAR-3 and anti-gp135 antibodies. (D) Quantification of cells with cell peripheral gp135 staining in C. Notice that the overexpression of PAR-3 increased the number of cells with peripheral gp135 staining not only in control cells but also in PAR-3 kd cells. Each photograph represents the projected views of optical sections from the apical to the basal membranes of cells. Bars, 20 mm.

• Supplemental Figure 3 -

  Fig. S3. mLgl knockdown suppresses disassembly of apical junctions in response to Ca²⁺ depletion. (A) Confluent monolayer of control (21-1) or mLgl kd (24-15) cells were incubated with LC medium for the indicated time. The cells were fixed, permeabilized and stained with anti-ZO-1. (B) Each clone incubated with LC medium for 5 hours was stained with anti-occludin together with anti-ZO-1. Notice that disappearance of apical junction proteins from cell-cell junctions was suppressed in mLgl kd cells. Each photograph represents the projected views of optical sections from the apical to the basal membranes of cells. Bars, 20 mm.

• Supplemental Figure 4 -

  Fig. S4. Overexpression of PAR-3 induces formation of cell aggregates without lumen during cystogenesis. (A) MDCK cells with and without T7-PAR-3 expression were cultured in collagen gel for 7 days. Then, the cells were fixed and costained with gp135 and T7 antibodies. Cell nuclei were stained with TOPRO3. (B) Quantification of abnormal cell aggregates in total cysts. Values are means (ąs.d.) of three independent experiments. Notice that PAR-3 overexpression increases the ratio of abnormal cell aggregates. Each photograph represents the projected views of optical sections from the apical to basal membrane of cells. Bars, 20 mm.

• Supplemental Figure 5 -

  Fig. S5. The effects mLgl kd, PAR-3 kd or PAR-3 overexpression on E-cadherin localization in depolarized MDCK cells. (A, B) Confluent monolayer of mLgl kd (A) or PAR-3 kd (B) cells on permeable filter were incubated with LC medium for 20 hours, after which cells were fixed and sainted with anti-E-cadherin antibody. (C) Control cells (1-5) cultured on permeable filters were infected with adenovirus expression vectors encoding LacZ or T7-PAR-3 for 2 hours. The cells were then incubated with LC medium for 18 hours, after which cells were fixed and sainted with anti-E-cadherin antibody. Notice that E-cadherin accumulation in intracellular region was suppressed in mLgl kd cells, whereas clear effect was not observed in PAR-3 kd cells or PAR-3 overexpressing cells.