Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1A-F - Growth rates are not altered in APC-deficient cells.
  (A) Fibroblasts constitutively lacking APC or (B) Fibroblasts before or after treatment with Cre recombinase, or (C) U2OS cells treated with control siRNA or APC-targeting siRNA were seeded at identical densities and cell numbers determined at indicated time points. (D) Wildtype (wt) and mutant (mut) or (E) floxed Fibroblasts before (ctrl) or after (cre) treatment with Cre recombinase and (F) U2OS cells treated with control or APC-targeting siRNA were grown to confluency. A scratch was introduced into the monolayer as in cells as shown in the migration experiments in Figure 1. The medium was supplemented with BrdU immediately after the scratch had been made or after cells had migrated for 24 hours. Cells were then fixed two hours later and probed with an anti-BrdU antibody. The proportion of cells in the migrating margin that had incorporated BrdU was determined by counting BrdU-positive cells using immunofluorescence. There was no difference in the number of proliferating cells in the migrating margins between the control and APC-depleted cells when 500-1,000 cells were examined in each case.
• Supplemental Figure 1G - (G) Lysates from the indicated cells were probed with antibodies against APC and GAPDH shown in Fig. 1. Using a LiCor Odyssey system the amount of APC relative to GAPDH in each sample was measured to confirm a reduction of APC between 96 and 80%.
  Method: BrdU incorporation
  Cells were grown to confluency and a scratch made to introduce a gap into the monolayer. The media was supplemented with 10nm BrdU immediately after the scratch was made or 24 hours later for 2 hours. Cells were fixed with methanol for 5 min –20°. DNA was denatured with 2N HCl for 15-20 min at 37° C, neutralized with 0.1M borate buffer pH8.5 that was changed twice over 10 min. Slides were placed in a humidified chamber and incubated with primary antibody for 60 min at room temperature (anti BrdU (ab 1893, Abcam 1:250) washed again with PBS and incubated with anti-sheep FITC (1;200, Jackson) and stained with DAPI as described before. The proportion of BRdU positive cells in the margin facing the scratch was determined.
• Supplemental Figure 2 - Lack of APC results in fewer nocodazole-resistant microtubules.
  (A) Fibroblast constitutively lacking APC (APC-mutant), wild type fibroblasts (APC-wt); (B) mutant fibroblasts before (floxed-ctrl) or after (floxed-cre) treatment with Cre-recombinase and (C) U2OS cells depleted of APC by RNAi were treated with nocodazole (fibroblasts) or vinorelbine (U2OS) to depolymerise microtubules. Immunostaining with antibodies against acetylated (acet-MTs) and unmodified microtubules showed that APC-deficient cells contained fewer stable microtubules and that the microtubules that were stabilized were usually acetylated
  Method:
  Transiently transfected PTK2 cells were placed into 4°C for 30 min. Cold media was replaced with media containing 10μg/ ml nocodazole and cells were incubated at 37° C for a further 75 min (PTK2). U2OS cells were treated with vinorelbine at 0.01ng/ml for three hours and fibroblasts were kept at 4°C for 30 min before receiving 10μg/ml nocodazole at 37°C for 30 minutes. Cells were then prepared for immunofluorescence as described above (Suppl. Fig. 2).
• Supplemental Figure 3 - APC depletion does not alter levels of stathmin/OP18 or KAP3.
  Cells lysates from fibroblasts constitutively lacking APC (APC-mutant) or fibroblasts before or after treatment with Cre-recombinase or U2OS cells after APC inactivation by RNAi and corresponding controls were blotted and probed with antibody against KAP3, APC, stathmin/OP18, and tubulin as a loading control.
• Supplemental Figure 4 - Expression of GFP-APC
  (A) Cell lysates of SW480 cells transiently expressing the indicated APC constructs were probed with antibodies against C-terminal APC (top panel) or GFP (bottom panel) to confirm expression of the different APC proteins. (B) PTK2 cells were transfected with GFP, GFP-APC full length, or GFP-C-APC. After 48 hours, cells were harvested and GFP fluorescence was measured using FACS. Although more cells expressed GFP and the average amount of GFP per cell was higher than in APC or C-APC transfected cells, there was no difference in either of these parameters between APC and C-APC expressing cells. This confirms that the transfection efficiency and, more importantly, the amount of expressed APC protein per cell was similar in these cases.
• Supplemental Figure 5 - Expression of full length APC leads to more highly asymmetrical cells.
  These data are the same as shown in Figure 4 in the manuscript. However, the data are represented as a histogram to illustrate the distribution pattern of the actual length/width ratios. This illustrates that the number of extremely asymmetrical cells increases when full length APC is expressed but is also shows that the distribution of these values is not “normal” and thus requires a statistical analysis that deals with non-normal distributions like the Kruskal-Wallis One Way Analysis of Variance of Ranks as cited.