JCS02679 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. APC but not b-catenin in cortical clusters rapidly increases in response to NGF. PC12 cells plated for 48 hours on collagen-coated coverslips were treated for the indicated hours (0, 2, 4, 6) with NGF, methanol-fixed as described (Materials and Methods) and co-immunolabeled for APC and b-catenin (see Materials and Methods for antibodies used). Images were taken as described, with the same exposure times for each immunostain and brightness/contrast was equally adjusted for each immunostain with Adobe Photoshop. APC fluorescence intensity in cortical clusters (arrowheads in left column) rapidly increased upon stimulation with NGF whereas b-catenin fluorescence intensity in the cortical APC clusters did not significantly change (arrowheads in right column). Bar, 5 mm.

• Supplemental Figure 2 -

  Fig. S2. b-catenin-depleted cells are more elongated.(A) A pool of siRNAs specifically targeting b-catenin (obtained from Dharmacon) was transfected in HeLa cells, which were fixed and stained after 48 hours. HeLa cells depleted for b-catenin by siRNAs (second column) are often more elongated than cells treated with control siRNA against GFP (first column). Top row shows immunolabeling for b-catenin (with same exposure time, enhancement and magnification), and bottom row shows co-immunolabeling for APC (see Materials and Methods for antibodies used). Note in bottom right panel that APC clusters form at tips of cells (arrowheads) in absence of b-catenin. (B) Elongation was quantified in ImageJ as the major axis of an ellipse fit to the cell. b-catenin-depleted HeLa cells have a significantly longer major axis than control siRNA-treated cells. (Shown is the average±s.e.m. of three independent experiments with 82–113 noncontacting, mononuclear cells counted in each experiment. *P<0.05 using Student’s t-test)