Polarity proteins PAR6 and aPKC regulate cell death through GSK-3 in 3D epithelial morphogenesis

JCS007443 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Apoptotic phenotype by PAR6ΔN overexpression is dose dependent. (A) Cysts from wild-type MDCK and three clones overexpressing different levels of Myc-PAR6ΔN were subjected to immunoblot with antibodies against Myc and PAR6B. (B) Percentage of cysts indicating lacking cleaved caspase-3 staining was quantified in wild type MDCK and 3 different clones overexpressing Myc-PAR6ΔN. n=300 cysts per clone.

• Supplemental Figure S2 -

  Fig. S2. PAR6ΔN-induced apoptosis is caspase-dependent. PAR6ΔN cysts were treated with either a carrier (DMSO) or caspase inhibitor, zVAD-fmk (50 μM) three times at 0, 4 and 6 days and apoptosis was measured using the In Situ Cell Death Detection kit (Roche). Fluorescein labels incorporated in nucleotide polymers were detected by fluorescence microscopy in the range of 515-565 nm. Bars, 100 μm.

• Supplemental Figure S3 -

  Fig. S3. PAR6ΔN cysts were more proliferative than WT MDCK. (A) Wild-type MDCK and Myc-PAR6ΔN cells grown on collagen for 7 days were fixed, permeablized and immunostained for cleaved caspase-3 (green), Ki-67 (red) and actin (white), Bar, 10 μm. (B) The levels of proliferation were measured using a CyQUANT cell proliferation kit. Mean and s.d. of triplicates are shown.

• Supplemental Figure S4 -

  Fig. S4. GFP-PAR6B and GFP-PAR6ΔN are partially localized at the apical region. GFP-PAR6B or GFP-PAR6ΔN was transfected into MDCK cells and their localizations observed in live cells. The right panel shows a merged image with differential interference contrast (DIC). Bar, 10 μm.