GTSE1 expression in breast cancer tumors and cells correlates with time to metastasis and invasiveness.

<p>(A) Kaplan–Meier survival curve of time to distant metastasis of breast cancer patients classified according to the expression of GTSE1. Red line: cases with high expression of GTSE1, blue line: cases with low expression of GTSE1. (p-value <10?–15) (B) Boxplots of the distribution of gene expression intensities of GTSE1 across different breast cancer subtypes (Grade 1, 2 or 3; p<10?-5; linear regression analysis),. (C) Western blot analysis of GTSE1 and EB1 protein levels in different breast cancer cell lines. Tumor types are: F, fibrocystic disease, non-transformed, immortal cell line; IDC, invasive ductal carcinoma; AC, adenocarcinoma; MC, metaplastic carcinoma. Invasive potential is characterized as not invasive (−), invasive (+), or highly invasive (++). Adapted from Neve et al. Cancer Cell 2006. (D) Quantitative RT-PCR analysis of GTSE1 and EB1 relative mRNA levels in MCF7 and MDA-MB-231 cells. Error bars represent the standard error of the mean from three independent experiments. p<0.01 (Student’s t-test). (E) Transwell migration assay and western blot of the MDA-MB-231 cell line. Cells were transfected with control (CON) or GTSE1 siRNA for 36 hours, trypsinized, and seeded on transwell membranes. Histograms show the mean number of cells/area that migrated through the transwell after 16 h (10 replicates/experiment). Error bars represent the standard error of the mean from three independent experiments. * indicates p<0.05 (Student’s t-test). Western blots were performed on cells after the same treatment, and blotted with anti-GTSE1 and anti-actin. (F) Transwell migration assay and western blot of the MCF7 cell line containing a stably integrated GTSE1 overexpression construct (pBABE-GTSE1) or empty vector (pBABE). Cells were trypsinized and seeded on transwell membranes. Histograms show the mean number of cells/area that migrated through the transwell after 16 h (10 replicates/experiment). Western blots were performed on cells after the same treatment, and blotted with anti-GTSE1 and anti-actin.</p

GTSE1 modulates cell migration in an EB1-dependent manner.

<p>(A) Transwell migration assay and western blot in U2OS cells, and U2OS cells stably expressing RNAi-resistant wild-type GTSE1-GFP (GTSE1^WT₂₀₄), or RNAi-resistant GTSE1-GFP mutated at the SKIP motifs (GTSE1^Sk₂₀₂). Cells were transfected with control (CON) or GTSE1 siRNA for 36 h and seeded on transwell membranes. Histograms show the mean number of cells/area that migrated through the transwell after 16 h (10 replicates/experiment). Error bars represent the standard error of the mean from three independent experiments. Western blots were performed on cells after the same treatment, and blotted with anti-GTSE1 and anti-actin. (B) Transwell migration assay and western blot in Wi38 cells. Cells were transfected and analysed as in (a). (C) Transwell migration assay and western blot in HCT116 wild type, HCT116 p53−/−, and HCT116 p21−/− cell lines. Cells were transfected and analyzed as in (a). Western blots were performed on cells after the same treatment, and blotted with anti-GTSE1, anti-p21, anti-p53, and anti-actin. (D) Transwell migration assay and western blot in H1299 cells containing inducible constructs for expression of wild type GTSE1 (GTSE1 WT) or SKIP-domain mutated GTSE1 (GTSE1 Sk). Cells were untreated (−) or treated with Ponasterone A (PonA) (+) for 24 h to induce GTSE1 expression, then trypsinized and seeded on transwell membranes. Histograms show the mean number of cells/area that migrated through the transwell after 16 h (10 replicates/experiment). Western blots were performed on cells after the same treatment, and blotted with anti-GTSE1 and anti-actin.</p

GTSE1 is an EB1-dependent microtubule plus end growth tracking protein.

<p>(A) Still images of a live U2OS cell stably expressing GTSE1-GFP and mCherry-alpha-tubulin from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051259#pone.0051259.s007" target="_blank">Movie S1</a>. GTSE1-GFP is enriched at growing microtubule plus ends, and also associated with the microtubule lattice. (B) Western blot showing GTSE1 and EB1 levels after EB1 RNAi. U2OS cells stably expressing GFP-GTSE1 were transfected with a control (siCONT) or EB1 (siEB1) siRNA for 36 h. Blots were probed with antibodies against GFP, EB1 or actin (loading control). (C) Still images of live U2OS cells expressing GTSE1-GFP after transfection with control (CON) or EB1 siRNA, from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051259#pone.0051259.s008" target="_blank">Movies S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051259#pone.0051259.s009" target="_blank">S3</a>. After EB1 depletion, GTSE1-GFP no longer tracks growing microtubule ends, but remains associated with the microtubule lattice.</p

GTSE1 is recruited to microtubule plus ends through short EB1-interaction motifs.

<p>(A) Sequence alignment of hGTSE1 amino acids 503–538 that contain tandem conserved SKIP-like motifs. The first four rows contain GTSE1 homologs from human (hs), mouse (mm), Xenopus (xl) and zebrafish (dr). The last three rows show conserved regions from other human +TIPs. SKIP-like motifs are highlighted in green boxes. Conserved TP motifs are highlighted in grey boxes. Basic residues are colored red, serines and threonines are colored blue. (B) GTSE1 immunoprecipitates EB1 in U2OS cells. U2OS cell lysates were immunoprecipitated with anti-GTSE1 antibody, or anti-GFP as a control. Input lysate and immunoprecipitated fractions were run by SDS-PAGE and Western blotted with either anti-GTSE1 or anti-EB1 antibody. (C) <i>In vitro</i> pull-down binding assay using purified GST or GST-EB1 fusion proteins incubated with <i>in vitro</i> translated ³⁵S-labeled GTSE1 WT (hGTSE1 WT IVT) or GTSE1 mutated at the SKIP motifs (L511N P512N L522N P523N) (GTSE1 Sk IVT). Inputs represent 20% of IVTs used for pull-down assays. The top gel shows IVT GTSE1 by autoradiograph, bottom gels are commassie stained. GST-EB1 interacts with <i>in vitro</i> translated GTSE1, but not GTSE1 mutated at SKIP motifs. (D) Still images of live clonal U2OS cells expressing wild type GTSE1-GFP (GTSE1^WT₂₀₄) or GTSE1-GFP mutated at the SKIP motifs (L511N P512N L522N P523N) (GTSE1^Sk₂₀₂)(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051259#pone.0051259.s010" target="_blank">Movie S4</a>). Similar to EB1 depletion, the mutated GTSE1-GFP does not track growing microtubule tips, but localizes to the microtubule lattice. All scale bars represent 10 microns.</p

Differentially expressed genes presenting AS events in the striatum tissue of KI mouse models.

The bar graph presents the number of differentially expressed genes (DEG) which also are characterized by at least one significant AS event in the striatum of KI animal models of HD. Logarithmic fold change (LogFC) of their expression compared to Q20 control is reported in the y-axis. Transcripts are filtered based on significant p-value (p-value (TIFF)</p

Neuronal differentiation impact on small RNAs.

A) The coloured bar graphs report the number of small RNAs (>1 count/sample, see also Methods) comparing ESC and NPC conditions. Series of 4 Htt CAG expansion alleles (Q20, Q50, Q92 and Q111) are presented. Different classes of small RNAs are examined. Abbreviations as follows: microRNAs (miRNAs), Mitochondrial transfer RNAs (Mt-tRNAs), processed pseudogenes, ribosomal RNAs (rRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs) small Cajal body-specific RNAs (scaRNAs), To be Experimentally Confirmed (TEC), transcribed processed pseudogenes. B) The heatmap presents the number of expressed (> 1 count per million, CPM) small RNAs among heterozygous Htt CAG knock-in HttQ20, HttQ50, HttQ92 and HttQ111 (Q20, Q50, Q92, Q111), comparing ESC and NPC lines. Different classes of small RNAs are examined. Abbreviations as in GENCODE transcript biotypes [86]. Color code bar (upper right) reports the number of expressed small RNAs in each condition. C) The bar chart shows the number of small RNAs differentially expressed between Htt Q111 versus Q20 genotypes. The comparison is presented for pluripotent (ESC) and neural committed progenitors (NPC). The number of small RNAs increasing (Increasing in Q111—upper part of the plot), and decreasing their expression in Q111 versus Q20 (Decreasing in Q111—lower part of the plot) is depicted. (TIFF)</p

Aberrant linear alternative splicing in the striatum of KI animal models of HD.

A) Bar graphs show the number of differential AS events in the striatum and cortex from 6 mouse KI models (Q20, Q80, Q92, Q111, Q140 and Q175) of HD, presenting different Htt CAG repeat lengths and 3 ages (2, 6, and 10 months). The number of events is shown for each genotype, time point and brain region. The inclusion level is calculated in comparison to Q20 controls and the positive or negative values are plotted. Source data by Langfelder P. et al (2016) [28]. Further details can be found in the Methods section. Each color of the bar chart represents a different AS type. B) Circos plot represents the number of transcripts within the striatum—showing differential AS events—shared between different genotypes (Q80, Q92, Q111, Q140 and Q175) and time points (2, 6, and 10 months). Conditions (genotypes and/or time points) sharing more than 50 transcripts are depicted in red. C) Weighted nodes graphical representation shows the functional enrichment analysis for transcripts displaying significant skipped exon (SE) events in the striatum. Highly expanded Htt CAG sizes (Q140 and Q175), the major contributors to aberrant SE in the striatum, are shown separated. Nodes’ size legend is depicted at the bottom. D) Representative agarose gel images (left) and quantification plots (right) report the RT-PCR results of AS validation for Crem, Adamts6, and Trpm4, selected transcript targets. RT-PCR assay and quantification were performed on striata RNA from an independent set of wild-type (WT), Q20 and Q111/Q175 mice. Transcripts isoforms with inclusion or exclusion of the variable exon (in yellow) are visualized and quantified. The plots report the PSI, percent-spliced-in. *p-value n = 3), error bars indicate standard deviation.</p

<i>Htt</i> CAG size dictates a specific linear AS signature in neural cells partly leading to NMD.

A) The Venn diagram reports the comparison between striatal (STR) and neuronal progenitors (NPC) SE events. All genotypes (Q20, Q80, Q92, Q111, Q140 and Q175) and time points (2, 6 and 10 months) for striatal districts and all genotypes (Q20, Q50, Q92, Q111) for NPC were combined. Shared SE events (18,85% of the total striatal SE events, p-value: 0, Fisher’s exact test) are indicated in the intersection. B) The histogram reveals shared GO&KEGG pathway between SE and NPC events, mainly involved in ‘cell junction’ and ‘synapse organization’. Terms are ordered by −log10(p−value). The number of genes in each term/pathway is indicated (numbers within columns). C) The Venn diagrams show the proportion of SE events in the striatum (STR) and neuronal progenitors (NPC) also annotated as NMD. More than one third of SE events (36.1% in STR and 36.2% in NPC) is annotated as NMD. The p-value of enrichment for each intersection is indicated. The tables for striatum and NPC, highlight the transcriptional dysregulation of Upf1, and Smg1, important regulators of the NMD pathway. D) A smaller proportion of SE events (7.8% in STR and 8.4% in NPC) intersected with transcripts subjected to m6A RNA modification (see methods). E-F) Motif analysis identified the splicing factors and/or RBP binding sites in the ± 100bp upstream and downstream adjacent regions to the alternatively spliced exons for the striatum (E) and for NPC (F). The p-value of enrichment testing for individual motifs in each data set is indicated. The candidate binding splicing factor and/or RBP family is shown.</p

Related to Fig 1.

The spreadsheets (n = 5) report the total description of the alternative splicing (AS) events observed in 1. the striatum (STR), 2. the cortex (CTX) and 3. liver. All data are calculated comparing Htt CAG expanded genotypes (Q80, Q92, Q111, Q140, Q175) to Q20 control (see methods). The GO terms and pathways enrichment for 4. AS events in the STR and 5. Skipped exon (SE) events in the STR are presented. (XLSX)</p

<i>Htt</i> CAG length correlation with linear alternative splicing events in KI mouse models.

The bar graphs report the total number of differential AS events in the striatum (upper row, light blue), cortex (mid row, green) and liver (bottom, purple row) from mouse KI models of HD, presenting 6 different Htt CAG repeat lengths (Q20, Q80, Q92, Q111, Q140 and Q175) and 3 time points (2, 6, and 10 months). The inclusion level is calculated in comparison to Q20 controls and the positive or negative values are plotted in the graph. The number of events is reported for each genotype and time point (different Y-axis values are presented). Source data by Langfelder P. et al (2016) [28]. Further details can be found in the Methods section and S1 Table. The Pearson’s correlation (R2) between differential AS and Htt CAG expansion is plotted in each graph. Standard deviations and trend lines are presented. (TIFF)</p