Fig 2: Microinjection with EB1 or APC specific antibodies affects chromosomal congression at the metaphase plate.A: Movies of microinjected cells were examined and the last frame before anaphase onset identified. The distance separating the widest two points of the metaphase plate was then measured as indicated. In the representative examples shown it can be seen that the metaphase plate was more tightly compacted in cells injected with control IgG (panel a) than in cells microinjected with 1A11 (panel b). Scale bar = 5 µM. The red traces indicate the outline of the metaphase plates. B: Summary of the metaphase plate thickness data. The metaphase plate was significantly wider in cells microinjected with APC or EB1 specific antibodies when compared to cells injected with the non-specific antibody 4 U. Mean distances +/- SEM are shown. Significant results are shown with an asterisk. P values were for 1A11, p<0.005; ALI 12–28, p<0.005; C-APC 9.9, p<0.05 and C-APC 28, p<0.005. (4 U, n = 11; 1A11, n = 14; ALI 12–28, n = 11, C-APC 9.9, n = 12; C-APC 28.9, n = 7).

Fig 3: Microinjection of an inhibitory EB1 fragment into NRK-52E cells recapitulates phenotypes observed in EB1 antibody injected cells.Mitotic NRK-52E cells were microinjected with the dominant-negative EB1 fragment GST-EB1-C84 or with GST alone. A) Injection of low levels of GST-EB1-C84 inhibited progression through mitosis. B) Cytokinesis was significantly delayed in mitotic cells injected with GST-EB1-C84 that completed mitosis (p = 0.01). C) Chromosomal congression to the metaphase plate was significantly inhibited in cells microinjected with GST-EB1-C84. D) The majority of cells microinjected with GST-EB1-C84 had misaligned spindles at TP1, most of which had not corrected by the time cells reached telophase. E) Some cells microinjected with GST-EB1-C84 showed severe asymmetry in spindle movement, as shown by a ratio value of <0.5. Different colours and shapes represent individual cells. (F) Cells injected with GST-EB1-C84 tended to possess spindles that were placed further from the cell centre, though this trend did not reach statistical significance. (G) A higher proportion of cells injected with GST-EB1-C84 exhibited cortical blebbing, though it was not mitotic stage specific.

Fig 4: Diagram showing the possible interactions of EB1 with main interactants and the effect of antibody binding.Blocking of EB1 with 1A11 enhances some interactions (CLIP-170, APC), reduces interactions with p150Glued and has no effect on other interactions, for example with MCAK (shifted balance after preincubation with specific antibody).

Fig 5: PI3K activity increases tubulin gene expression through autoregulation.(A) DGE in parental and cells expressing constitutively active PI3K H1047R mutant cells, treated with DMSO or indicated PI3K inhibitors for 4 h. (B and C) Relative expression of TUBA1A and TUBB unspliced pre-mRNA (B) and spliced TUBA1A and TUBB mRNA (C) in parental and PI3K mutant cell lines H1047R and E545K, treated with DMSO control or 1 µM GDC-0941 for 4 h. All the relative gene-expression data are normalized to housekeeping gene GAPDH or RPL19 and to DMSO-treated parental cells. (D) Tubulin partitioning to unpolymerized (soluble, S) and polymerized (P) normalized to loading control (GAPDH for S; HISH3 for P) and to DMSO-treated parental cells in parental and cells expressing constitutively active PI3K mutants H1047R and E545K, treated with DMSO or 1 µM GDC-0945 for 4 h. (E) DGE in A2058 cells treated with DMSO control or the indicated concentrations of PI3K inhibitors for 6 h (GEO series GSE66343 [28]). (F–G) RT-qPCR in A2058 cells treated with 1 µM GDC-0941 for indicated periods of time (x-axes). (H) Tubulin partitioning to unpolymerized (S) and polymerized (P) normalized to loading control (GAPDH for S; HISH3 for P) and to DMSO-treated cells in control and cells treated for 6 h with indicated 1 µM PI3K inhibitors. (I) Representative immunofluorescence images of control and cells treated with 1 µM GDC-0941 for 6 h and stained with anti-EB1 antibody (cyan), anti-ß-actin, and Hoechst (red). (J) Relative number of detected EB1 comets per cell area in DMSO control and A2058 cells treated with 1 µM GDC-0941 for 6 h, normalized to DMSO-treated cells (100–200 cells). (K) A proposed model describes the mechanism through which PI3K signaling regulates tubulin gene expression through modification of tubulin autoregulation. Bar plots in all panels represent average values, and error bars standard deviations from three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 in paired Student t test, in all panels. Black and white asterisk represent statistical significance relative to DMSO-treated parental cell line, green asterisks represent statistical significance relative to DMSO-treated control of the same cell line. a.u., arbitrary unit; DGE, differential gene expression; EB1, end-binding protein 1; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GEO, Gene Expression Omnibus; HISH3, histone H3; pAKT, phospho-AKT; PI3K, phosphatidylinositol-4,5-biphosphate 3-kinase; RPL19, ribosomal protein L19; RT-qPCR, reverse-transcription quantitative PCR; TUBA, a-tubulin; TUBB, ß-tubulin; TUBD, d-tubulin; TUBE, e-tubulin; TUBG, ?-tubulin; WT, wild type.