Journal: bioRxiv

Article Title: APC -mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

doi: 10.1101/2020.09.18.303016

Figure Lengend Snippet: ( a ) Immunostaining of AURKA, p-T288 AURKA (left), and microtubules (MTs) with Hoechst 33342 to stain DNA (left). See also Extended Data Table 1. Ratios of fluorescence intensities (phosphorylated vs. total protein) of AURKA (T288) and PLK1 (T210), and fluorescence intensities of γ-tubulin at centrosomes are shown (right). Scale bars: 5 μm. ( b ) Binding sites for centrosomal proteins and components of the β-catenin destruction complex. ( c ) In vitro AURKA autophosphorylation at T288 detected by western blotting. ( d ) In vitro kinase assay of AURKA autophosphorylation. ( e, f ) HEK293T cells were transfected with GFP-fused AURKA, AXIN1, β-catenin, and APC fragments (see Extended Data Figure 3a) as indicated and then subjected to western blotting using the indicated antibodies. APC bands are marked with asterisks. Note that the phosphorylated AURKA band always appeared as multiple bands and AXIN1 levels were increased when coexpressed with binding APC fragments. For uncropped versions of blots, see Extended Data Table 2. **P < 0.01; ***P < 0.001, Student’s t-test (a), Tukey–Kramer method (c, d)

Article Snippet: Briefly, inactive AURKA (0.3 pmol; SignalChem) was mixed with various concentrations (0.5-2 pmol) of purified GST, Axin1 (OriGene Technologies) or TPX2 (SignalChem) for 10 min at RT.

Techniques: Immunostaining, Staining, Fluorescence, Binding Assay, In Vitro, Western Blot, Kinase Assay, Transfection

Journal: bioRxiv

Article Title: APC -mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

doi: 10.1101/2020.09.18.303016

Figure Lengend Snippet: ( a, b ) Domain structures of APC and AXIN1, and the analysed fragments. Amino acid numbering is based on human APC transcript variant 3 (NM_000038) and human AXIN1 transcript variant 1 (NM_003502). Associations of APC with AURKA and AXIN1 were assessed by in-cell colocalization analysis using a series of deletion mutants. The results are shown on the right. ( c ) Direct binding between APC fragments and AURKA/AXIN1/ch-TOG predicted in (a) was confirmed by in vitro pull-down assays using purified proteins. The APC arm and APC-C6 bound to AURKA. The APC arm also bound to the C-terminal DIX domain of AXIN1. XMAP215 , the Xenopus homologue of ch-TOG, bound to APC-C3. We used Xenopus XMAP215 because we could not purify full-length human ch-TOG from E. coli . The C-terminal one-third bound to the APC arm. See also Extended Data Table 2. ( d ) Yeast two-hybrid screening using the APC arm region as bait identified pericentrin. The two identified pericentrin clones are shown.

Article Snippet: Briefly, inactive AURKA (0.3 pmol; SignalChem) was mixed with various concentrations (0.5-2 pmol) of purified GST, Axin1 (OriGene Technologies) or TPX2 (SignalChem) for 10 min at RT.

Techniques: Variant Assay, Binding Assay, In Vitro, Purification, Two Hybrid Screening, Clone Assay

Journal: bioRxiv

Article Title: APC -mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

doi: 10.1101/2020.09.18.303016

Figure Lengend Snippet: ( a ) AXIN1 structure and AURKA-activating region that also binds to β-catenin . ( b ) Effects of AXIN1 on AURKA phosphorylation were analysed using the HEK293T cell overexpression assay system. HEK293T cells were transfected with GFP-fused AURKA and full-length AXIN1 or fragments, lysed, and then subjected to western blot analysis using anti-GFP and anti-p-T288 AURKA antibodies. The left panel is same with . See also Extended Data Table 2. ( c, d ) Analysis of primary MEFs from wildtype (WT) and Apc 1638T mice. Immunostaining of microtubules (MTs), γ-tubulin, AURKA, and p-T288 AURKA (c). Immunostaining intensity of γ-tubulin, total AURKA protein levels (t-AURKA), autophosphorylated AURKA levels (p-AURKA), and the autophosphorylation ratio (p-AURKA/t-AURKA ratio) were normalized to those of the wildtype, which were set to 1.0 (d). Scale bars: 5 μm. ***P < 0.001; Student’s t-test.

Article Snippet: Briefly, inactive AURKA (0.3 pmol; SignalChem) was mixed with various concentrations (0.5-2 pmol) of purified GST, Axin1 (OriGene Technologies) or TPX2 (SignalChem) for 10 min at RT.

Techniques: Over Expression, Transfection, Western Blot, Immunostaining

Journal: bioRxiv

Article Title: APC -mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

doi: 10.1101/2020.09.18.303016

Figure Lengend Snippet: ( a ) AURKA status at centrosomes obtained from immunofluorescence images. See also Extended Data Table 1. Total AURKA protein levels (t-AURKA), autophosphorylated AURKA levels (p-AURKA), the auto-phosphorylation ratio (p-AURKA/t-AURKA ratio), and mitotic error rate in parental MCF10A cells that expressed full-length APC, APC883 cells, and cell lines derived from APC883 cells are shown as indicated. Mitotic errors were monitored by generation of lagging chromosomes (lower right). ( b ) Representative images of immunostained MCF10A cells that expressed myc-WTβ-catenin or myc-MMβ-catenin (left) and signal intensity analysis of AURKA (right). ( c ) FRAP analysis of GFP-AURKA in cells that expressed WT β-catenin or MMβ-catenin. Representative images (left) and average FRAP recovery half-time (right) are shown. See also Extended Data Table 1. ( d ) Duration of mitosis in each cell line shown as a cumulative histogram. See also Movies 4–6 and Extended Data Table 1. ( e ) Schematic diagram of AURKA regulation by the APC/AXIN1/β-catenin complex. Scale bars: 5 μm. For (a), ***P < 0.001 vs. MCF10A, ### P < 0.001 vs. APC883, Student’s t-test. ***P < 0.001, Student’s t-test (b, c), Tukey–Kramer method (a, d).

Article Snippet: Briefly, inactive AURKA (0.3 pmol; SignalChem) was mixed with various concentrations (0.5-2 pmol) of purified GST, Axin1 (OriGene Technologies) or TPX2 (SignalChem) for 10 min at RT.

Techniques: Immunofluorescence, Derivative Assay