Table S2 from The Ndc80 complex targets Bod1 to human mitotic kinetochores

Kinetochore and centromeric proteins detected as interactors of Bod1-GFP in mitotic HeLa cell

Supplementary figures S1-S5 from The Ndc80 complex targets Bod1 to human mitotic kinetochores

Supplementary figures and associated figure legend

Functional consequences of the absence of BOD1 in patient-derived fibroblasts.

<p>(A) Flow cytometric analysis of cell cycle profile in WT and <i>BOD1</i>^-/- primary fibroblasts electroporated with control or <i>BOD1</i> siRNA. Error bars represent standard deviation. (B) Immunoblotting of BOD1 and tubulin from cell lysates simultaneously electroporated with samples analysed in (A). (C) Representative DIC timelapse imaging of primary fibroblast cells undergoing mitosis. Nuclear Envelope Breakdown (NEB) and Anaphase Onset (AO) are indicated. (D) Cumulative timing of NEB to AO timing in Primary Fibroblast cell lines. Error bars represent standard deviation. P<0.001 for <i>BOD1</i>^-/- cells to each WT sample. Insufficient data collected for <i>BOD1</i>^-/- cells to determine statistical significance. (E) Immunofluorescence localization of PP2A-B56 in WT and <i>BOD1</i>^-/- Primary fibroblasts. DAPI (blue), centromeres (detected with ACA) (green), anti-PP2A-B56α (red). (F) Mean B56α levels at kinetochores of WT and <i>BOD1</i>^-/- Primary fibroblasts (P<0.001). Error bars represent SEM. (G) Immunofluorescence localization of PLK1 in WT and <i>BOD1</i>^-/- Primary fibroblasts. DAPI (white), anti-PLK1 (green), ACA (blue). (H-J) Mean PLK1 levels at kinetochores and centrosomes of WT and <i>BOD1</i>^-/- Primary fibroblasts, respectively (P<0.001 in each instance). Error bars represent SEM. (K) Immunoblotting of PLK1, BOD1 and tubulin in asynchronous WT and <i>BOD1</i>^-/- primary fibroblasts. (L) Immunoblotting of PLK1, BOD1 and tubulin in asynchronous and Monastrol arrested WT and <i>BOD1</i>^-/- Primary fibroblasts. (M) Immunofluorescence localization of Bod1 in WT Primary Fibroblasts. DAPI (white), ACA (blue), anti-Plk1 (red), anti-Bod1 (green). Scale = 5 μm. Inset shows a single bioriented kinetochore pair. (N) Immunoblotting of PP2A-B56δ, PLK1 and tubulin in WT primary fibroblast electroporated with indicated combinations of CTR, B56-pool or <i>BOD1</i> siRNA. Rescue of WT primary fibroblasts after siRNA depletion of Bod1 with plasmids expressing GFP fused to either siRNA resistant WT Bod1 or Bod1^T95E. (O) Mitotic profile of WT primary fibroblasts and <i>BOD1</i>^-/- fibroblasts 1 hr after release from RO 3306 into the indicated concentrations of BI 2536. Results show average of three independent experiments. A minimum of 100 mitotic cells counted per condition per experiment. Error bars represent SEM.</p

Expression of BOD1 and PLK1 in human tissues.

<p>(A) BOD1-specific quantitative RT-PCR experiments were carried out in triplicates, using RNA from the indicated tissues. All splice variants (indicated by the respective exon combinations) were investigated. Error bars represent the SEM. (B) Expression levels i.e. reads per kilobase of transcript per million reads mapped (RPKM), corresponding to BOD1 (NM_138369.2) and PLK1 (NM_005030.5) obtained by RNA-Sequencing of commercially available RNA-samples from different brain tissues, induced pluripotent stem cells (IPSC) and human embryonic stem cells (hES).</p

Presynaptic localisation of BOD1 in murine corticoneuronal cells.

<p>Representative indirect immunofluorescence confocal image (LSM510) of mouse cortical neurons transfected at day 7 after preparation with 0.1μg BOD1-GFP for 7hrs. Arrows indicate co-localization of BOD1-GFP (in green) with the (pre)synaptic marker anti-Bassoon (red). Insets are magnifications of the boxed area. The range indicator (RI) shows that the images are not overexposed.</p

Neuronal knockdown of Drosophila Bod1 affects learning and synapse development.

<p>(A-B') Knockdown of <i>Drosophila</i> Bod1 using the postmitotic, pan-neuronal promoter elav-Gal4 and three inducible RNAi lines affects non-associative learning in the light-off jump habituation paradigm. Jump responses were induced by repeated light-off pulses for 100 trials with a 1s inter-trial interval. Bod1 knockdown flies of genotypes (A) UAS-Bod1^vdrc105166/2xGMR-wIR; elav-Gal4, UAS-Dicer2/+, plotted as red squares, (B) UAS-Bod1^vdrc27445/2xGMR-wIR; elav-Gal4, UAS-Dicer2/+, plotted as blue squares, and (C) 2xGMR-wIR/+; UAS-Bod1^HMS00720/elav-Gal4, UAS-Dicer2, plotted as green squares, failed to habituate, i.e. to efficiently reduce their jump response upon repeated stimulation. The genetic background controls, generated by crossing the driver line to the respective genetic background of the RNAi line, are shown as grey circles (2xGMR-wIR/+; elav-Gal4, UAS-Dicer2/+). (A', B', C’) Quantification of average jump responses revealed that all three mutant genotypes habituated significantly slower (*** p<0,001). Red bar in (A') Bod1^vdrc105166, TTC = 49.75, n = 143 versus controls: TTC = 20.88, n = 134. Blue bar in (B'): Bod1^vdrc27445, TTC = 61.92, n = 93 versus controls TTC = 28.93, n = 87. Green bar in (C)’ Bod1^HMS00720, TTC = 10.03, n = 70 versus controls TTC = 5.51, n = 68. (D) Knockdown of <i>Drosophila</i> Bod1 using the elav-Gal4 promoter and RNAi lines Bod1^vdrc27445 and Bod1^vdrc105166 consistently affects synaptic branching at the <i>Drosophila</i> Neuromuscular Junction (see text). L3 muscle 4 synapses were labelled with anti-dlg1 and quantified using an in house-developed macro. A Bod1^vdrc27445 (UAS-Bod1^vdrc27445/2xGMR-wIR; elav-Gal4, UAS-Dicer2/+) and control (2xGMR-wIR/+; elav-Gal4, UAS-Dicer2/+) synapse is shown. Top panel in red: dlg1 labelling; bottom panels show the macro-annotated, quantified synapse). Asterisks highlight the increased number of synaptic branching points at the mutant synaptic terminal.</p

Nonsense Mutation in <i>BOD1</i> co-segregates with Intellectual Disability and leads to loss of BOD1 in patient tissues.

<p>(A) Family pedigree and co-segregation of the mutation within the family. Filled symbols represent affected individuals. Sequence chromatograms from one patient (V:2) and one parent (IV:1) are shown on the upper right. (B) Schematic representation of BOD1 (black) and the exon composition in alternative transcripts. Previously unknown transcripts are shown in green. Arrows indicate the location of primers used for RT-PCR experiments (C) Agarose Gel electrophoresis results of RT-PCR experiment. (D) qRT-PCR was performed on patient and control Fibroblasts. The experiments were performed twice with independent cells, each time in triplicate (Error bars represent the SEM). One representative result is shown. (E) NMD analyses of patient fibroblasts were performed twice with independent cell samples, each time in triplicate. The results are from pooled patient (<i>BOD1</i>^-/-) and control (WT) samples. CHX: cycloheximide, DMSO:Dimethyl sulfoxide. Error bars represent the SEM. (F) Western blot of protein extracts from fibroblast cells using a Bod1 polyclonal antibody. The Bod1 antibody recognizes a 22KDa protein, matching the full-length Bod1 protein. Alpha tubulin was used as a loading control.</p