JCS016246 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Akt2 is expressed much later than the early T567 ezrin phosphorylation. Frozen sections of mouse jejunum were stained with anti-p-T567ezrin (A,C, red) and with anti-Akt2 (type specific) (B,D, green) antibodies, and counterstained with DAPI (blue). (A,B) villus tips; (C,D) longitudinal section of a crypt. Bars, 20 µm.

• Supplemental Figure S2 -

  Fig. S2. Baculovirus infection does not substantially change Sf9 cell shape and shows similar F-actin distribution than cells expressing h-ezrin alone. Sf9 cells were infected with an empty (wild-type) baculovirus for 24 hours, fixed, and processed with FITC-phalloidin (green) and anti-gp64 (viral coat protein) antibody (red). Infected cells (arrows) displayed a diffuse cytoplasmic distribution of F-actin. Non-confocal images show the entire cell, including filopodia at the surface of attachment to glass, which are not shown in the confocal sections in Fig. 2. Bar, 10 µm.

• Supplemental Figure S3 -

  Fig. S3. PKCι knockdown does not result in apoptosis. CACO-2 C2BBe cells were transduced with non-replicating lentivirus particles as described in Fig. 4. Cells were transduced with empty virus (A,C) or with lentiviral particles expressing anti-PKCι shRNA (B). A positive control for apoptosis was done by incubating the cells in 100 µM staurosporine overnight before the stain (C). After 8 days in culture, the cells were stained with Apopercentage dye (red). In untreated cultures, either with empty virus (A) or shRNA-producing virus (B), apopototic cells were rare, and were found at a rate of only one every several fields (included in A and B as internal positive controls), whereas many cells per field were positive after the treatment with staurosporine (C). Bar, 20 µm.

• Supplemental Figure S4 -

  Fig. S4. PKCι knockdown decreases the ratio of pT567/total ezrin signal and the slight decrease in ezrin is not a transcriptional effect. (A) Parallel samples from CACO-2 C2Bbe cells transduced with empty virus (filled circles) or anti-PKCι shRNA lentivirus particles (empty circles) were extracted at various times after seeding and analyzed by immunoblot with anti-pT567 and anti-ezrin antibodies as in Fig. 4. The ratios of densitometric measurements for each band sequentially reprobed with both antibodies were calculated. (B) Two cultures of CACO-2 C2Bbe cells transduced with empty virus (empty, red) and three independent cultures of the same cells transduced with anti-PKCι shRNA lentivirus particles (shRNA, green), each one in triplicate, were used to extract and purify RNA. The RNA from each culture was normalized by 18S RNA content and used in a reverse transcriptase quantitative PCR reaction with multi-exon ezrin-specific primers. The signal was normalized to a sixth culture and the ratios expressed as mean ± s.d.

• Supplemental Figure S5 -

  Fig. S5. PKCι knockdown results in disorganization of the apical F-actin cytoskeleton. CACO-2 C2BBe cells were transduced with non-replicating lentivirus particles carrying only a gene for puromycin resistance (empty, A,C,E) or an anti-PKCι shRNA sequence under a pol III promoter and the same puromycin-resistance gene (shRNA, B,D,F). The cells were cultured on filters for 9 days, fixed, and stained with fluorescent phalloidin. The images are confocal X-Y sections at the apical level (A,B), at the basal level, immediately next to the filter (C,D), and X-Z 3D reconstructions of an 8-voxel thick section of the stack shown with the apical side up (E,F). Bars, 10 µm.

• Supplemental Figure S6 -

  Fig. S6. Transient transfection with A120E PKCι does not result in cell death. (A-C) CACO-2 cells were grown on engraved glass coverslips and transfected with V5-tagged A120E PKCι mutant. After 24 hours, the cells were incubated in Apopercentage stain and photographed under transmitted light (A). The same cultures were immediately fixed and processed for V5 immunofluorescence (red, B,C). Fluorescence images were acquired without (B) or with (C) the overlapped phase image. Arrows indicate transfected cells and the arrowhead (A) an apoptotic cell. (D-G) CACO-2 cells were grown on filters and transfected with V5-tagged A120E PKCι mutant on day 3 of culture. After 24 hours, the cells were incubated in ice-cold PBS supplemented with 1 mg/ml Lucifer Yellow CH for 15 minutes (signal pseudocolored in green, E,G) and fixed in formaldehyde. Then, the cells were processed for immunofluorescence with anti-V5 tag antibody (red, D,F) and counterstained with DAPI (blue). Lucifer yellow fluorescence was collected in the green channel in confocal stacks. The images are X-Z 3D reconstructions of the stacks with the apical side up. V5-positive cells excluded Lucifer Yellow CH and rare necrotic cells that took up the dye were never V5 positive (F,G). (H-I) CACO-2 cells were grown in 25 cm² flasks and transfected with V5-tagged K274W (dominant negative, H) or A120E (constitutively active, I) PKCι mutants on day 4 of culture. After 24 hours, the medium of each culture was collected and centrifuged to pellet cells. The pellets were resuspended in 1 ml tissue culture medium, seeded on collagen-coated coverslips and incubated for another 24 hours. The cells were then fixed, processed for V5 signal (red) and counterstained with DAPI (blue). Few, non-transfected cells were observed from cultures transfected with K274W PKCι (H) as opposed to more abundant V5-positive cells from cultures transfected with A120E PKCι (I). Bars, 10 µm.