EMBO/SourceData huggingface dataset:
Figure 1B. Volcano plot showing DE 1hr after NB1 (top) and IMR32 (bottom) cell treatment with ALKAL2. Dashed lines show DE thresholds. Up-/downregulated genes indicated in blue. Six genes that are DE in both cell lines and sensitive to the ALK inhibitor lorlatinib are indicated and labelled in red.D. Temporal dynamics of ALKAL2-induced transcription of ARC, EGR1-3, FOS and FOSB in NB1 and IMR32 cells in the presence and absence of the lorlatinib, as indicated.E. Immunoblot validation of ALKAL2 induction of EGR1 and FOS at the protein level in NB1 cells. Cells were treated for 0, 1 and 6h in the presence and absence of lorlatinib as indicated.G. Immunoblot analysis of ALKAL2 induction of pSRF in NB1 cells. Cells were treated for 0 and 1h in the presence and absence of lorlatinib as indicated. RNA-Seq analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.
Figure 2B. Volcano plots showing differential protein expression 1hr and 24hr after ALKAL2 +/- lorlatinib stimulation as indicated. Dashed lines indicate differential expression thresholds. Differentially expressed proteins indicated in blue. Most pronounced responding proteins indicated in black and labelled.C, D. Immunoblot analysis of VGF protein in NB cells. (C) NB1 cells after 24h stimulation with ALKAL2 in the presence or absence of lorlatinib. (D) CLB-BAR and CLB-GE cells after 24h inhibition with lorlatinib.Dashed lines indicate differential expression thresholds. Differentially expressed proteins indicated in blue. Most pronounced responding proteins indicated in black and labelled. E. Volcano plots showing differential phosphorylation. Labelling colors as in (B).G. Graphical representation of FOXO3 dynamics, indicating S253 phosphorylation and total FOXO3 protein levels in response to ALKAL2 stimulation, in the presence or absence of lorlatinib.H-I. Immunoblot validation of FOXO3a and STAT3 in response to ALKAL2 stimulation in the presence or absence of lorlatinib as indicated. The slower migrating FOXO3a band in SDS-PAGE in (H) likely reflects FOXO3a phosphorylation that is not seen in the presence of lorlatinib. Proteomic analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.
Figure 3A. Hematoxylin and eosine staining of longitudinal sections of P9 pups at the central part of the left celiac ganglion. B. Quantification of the area of celiac ganglia cross-section. Largest sections from the central part of left celiac ganglions of different individuals were chosen for the analysis. **p<0.01, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD. C. Hyperplasia quantification in central sections of P9 left celiac ganglions shown as a percent of hyperplastic regions areas per ganglion cross-section. **p<0.01, ****p<0.0001, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD. D. Kaplan-Meier survival curve of mice resulting from intercrosses of Th-MYCN hemizygotes and Alk-F1178S heterozygote mice (p<0.0001; log-rank test). Wild type littermates were excluded.E. Gross appearance, hematoxylin and eosin as well as Ki67 stained sections of representative Th-MYCN and Alk-F1178S;Th-MYCN tumours. Scale bars indicate 250μm.F. Ki67 immunohistochemically staining of P9 celiac ganglia in mice of the indicated genotype (quantified in G). G. Quantification of hyperplastic areas (shown as percent of hyperplastic regions per ganglion cross-section) and Ki67 expression (shown as positive for Ki67 staining areas per total area of the section through the ganglion central part at P9). (*p<0.05, ***p<0.001, ****p<0.0001, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD).
Figure 4A. The oncogenic activity of MYCN is potentiated by overexpression of ALKAL2. Kaplan-Meier survival curves for Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice. Also shown are Rosa26_Alkal2N, Alk-F1178S and control (Ctrl) mice. Comparison of survival of Th-MYCN alone and Rosa26_Alkal2;Th-MYCN curves showed a significant difference (p=0.003; log-rank test).Tumours harvested from Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice express NB markers. Tumours from all three genotypes were large, in most cases filling the abdominal cavity (B).Dissection post-mortem revealed that the majority of Rosa26_Alkal2;Th-MYCN (18/20) and Alk-F1178S;Th-MYCN (7/10) tumours did not involve the adrenal glands (C).Histological examination of Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN tumours revealed positive staining for NCAM1, synaptophysin (SYP) and Chromogranin A (CGA). Scale bars indicate 100μm. (D)Histological examination of Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN tumours was confirmed for NCAM1 and SYP along with MYCN, ALK and ALKAL2 by immunoblotting (E). Immunoblots are representative of 3 independent technical analyses.F. Accumulated Kaplan-Meier survival curves are shown for all monitored Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice over time, estimating tumour penetrance (p<0.001; log-rank test).
Figure 5RNA-Seq based differential gene expression analysis of tumours arising in Rosa26_Alkal2;Th-MYCN (Alkal2) [n=6], Alk-F1178S;Th-MYCN (AlkF1174S) [n=6] and Th-MYCN (MYCN) mice [n=4]. See Table S4 for detailed results. A. Read coverage of the codon-optimised Alkal2 transgene, confirming Alkal2 expression in Alkal2 tumours. C-D. Volcano plot showing differential expression (DE) between Alkal2 and MYCN tumours. Differentially expressed genes are shown in blue (DE in Alkal2 tumours only) or black (DE in both Alkal2 and AlkF1174S tumours, as shown in (D)). Top ranked genes labelled. Dashed lines represent DE cut-offs.E. DE heatmap based on unsupervised hierarchical clustering of 400 DE genes (rows) and 16 samples (columns, as indicated on top). Sample color legend as in (A). Color key shown on top left.F. Boxplot showing Vgf expression in the 3 tumour types as indicated. Box plots indicate median values and lower/upper quartiles with whiskers extending to 1.5 times the interquartile range. P values calculated using Wald test as reported by DESeq2.G. Histological examination of Th-MYCN, Rosa26_Alkal2;Th-null and Alk-F1178S;Th-MYCN tumours revealing positive staining for VGF. Scale bars indicate 100μm.H. Kaplan-Meier relapse free (RF) and event free (EF) survival probability curves from two different NB cohorts, the Versteeg 88 cohort (upper panel) and the Kocak 649 cohort (lower panel), as derived from the R2 platform. Patients with higher VGF expression are highlighted in blue, whereas patients with lower expression are highlighted in red. The log-rank test P values are indicated.
Figure 6Murine NB cell lines were generated from tumours arising in Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) mice. A. Alkal2 expression in cells derived from Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) NB. Immunoblotting analysis for ALKAL2 and tubulin in the indicated mouse NB cell lines. Whole cell lysates (30μg) were analysed in each lane. B. The effect of increasing concentrations of brigatintib on cell confluence was analyzed by IncuCyte Live Cell Analysis of both Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) cell lines. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.C-D. Brigatinib suppressed tumour spheroid formation and spheroid viability. Cells (#3456 or #3540) were treated with brigatinib (0, 150nM) for 4d in ultra-low attachment plates. The spheroid number was analyzed by IncuCyte® Live Cell Analysis Systems. Data are presented as means ± SEM from three independent experiments. *p<0.05, two-tailed unpaired student t-test. Scale bar (C) is 200μm.E. Tumour spheroids formed from either Rosa26_Alkal2;Th-MYCN (#3540) or Alk-F1178S;Th-MYCN (#3456) were formed at indicated cell number in ultra-low attachment plates for 3d and followed by brigatinib (0, 150nM) for 10d. Inhibitor was re-fed every other day. Cell viability was determined by CellTiter-Glo 3D cell viability kit and data is presented as mean ± SEM from five independent experiments. *p<0.05, ****p<0.0001, two-tailed unpaired student t-test.F. Cell viability. Mouse tumour derived cell lines #3540 (Rosa26_Alkal2;Th-MYCN) and #3456 (Alk-F1178S;Th-MYCN) were treated with brigatinib (125nM and 500nM) and viability was evaluated by using a resazurin-based assay. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.G. Brigatinib treatment (0 or 150 nM) for 6h resulted in inhibition of ALK phosphorylation, and of activation of downstream signaling (ERK1/2), as well as MYCN expression. Cell lysates (Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456)) were immunoblotted with the indicated antibodies.
Figure 7A. Cells derived from tumours arising in Rosa26_Alkal2;Th-MYCN (#4953) and Alk-F1178S;Th-MYCN (#4938) mice are sensitive to both lorlatinib and brigatinib. The effect of increasing concentrations of each ALK TKI (as indicated) on cell confluence was analyzed by IncuCyte Live Cell Analysis. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.B. Tumour volume changes over time for Rosa26_Alkal2;Th-MYCN mice treated with lorlatinib (10 mg/kg; twice daily) or vehicle control. Tumour volume was measured by ultrasound on Day 0 and 7, and by direct measurement at Day 14. Day 0 (lorlatinib n=7, Ctrl n=6), Day 7 (lorlatinib n=2, Ctrl n=5) and Day 14 (lorlatinib n=7, Ctrl n=6). Data shown represents mean ± SD. **p<0.005; two-tailed unpaired student t-test.C. Rosa26_Alkal2;Th-MYCN animals treated with lorlatinib did not display any significant loss of body weight compared with vehicle controls. Data shown represents mean ± SD.D. Representative ultrasound images of tumours observed in Rosa26_Alkal2;Th-MYCN mice with annotated measurements at Day 0 and Day 7. Tumours arise in the retroperitoneal space ventral to the aorta, Ao.E. Representative MRI imaging of Rosa26_Alkal2;Th-MYCN tumours in response to lorlatinib at 4d and 14d.F. Rosa26_Alkal2;Th-MYCN tumours from lorlatinib or vehicle controls were analysed for pHis. A representative field of view for each tumour at 40x (175740μm2) were manually counted. Data shown represents mean ± 95% CI. p=0.0286; Mann-Whitney test.
Figure 1Schematic diagram of ATXN8OS (top strand) and ATXN8 (bottom strand). RNA-seq read coverage visualized by Integrative Genomic Viewer across the ATXN8/ATXN8OS locus is depicted.B Bar graph showing relative mRNA levels of ATXN8 and ATXN8OS. FPKM values for each transcript are normalized to FPKM value for ATXN8OS. (****; p<0.0001; mean ± SEM; unpaired t test) FPKM= fragments per kilo base of transcript per million mapped reads.D Immunohistochemistry of 10-12 month old end-stage SCA8 BAC mice brain tissue shows accumulation of novel polySer RAN protein (detected by unique antibody to the polySer protein C-terminus, α-SerCT) in cerebellar white matter, brainstem, hippocampus and layer II of the frontal cortex. Representative polySer aggregates are indicated by black arrows. No aggregates are found in age matched non-transgenic (NT) littermates (n=6 for each cohort).F Immunohistochemistry of human SCA8 autopsy tissue (n=4-7) shows accumulation of novel polySer RAN protein in cerebellar white matter, brainstem and frontal cortex.
Figure 2A Immunohistochemistry (IHC) of end-stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in the molecular layer and cerebellar white matter and that polyGln but not polySer accumulates in Purkinje cells. Representative polySer aggregates are indicated by black arrows. Inset: higher magnification of molecular layer and white matter.B IF double staining shows no co-localization of polyGln and polySer in frontal cortex, pons or hippocampus of end-stage SCA8 BAC mice.C IF double staining of end-stage SCA8 BAC frontal cortex shows exclusive localization of polyGln (mouse α-Gln, red, bottom panel) in neurons (rabbit α-NeuN, green, bottom panel). In contrast, polySer (Rabbit α-SerCT, red, top panel) shows widespread accumulation in the frontal cortex including within neurons (mouse α-NeuN, green, top panel).D IHC of SCA8 human cerebellum shows that PolySer accumulates in the white matter but not in the Purkinje cells (left panels) while polyGln accumulates in Purkinje cells but not cerebellar white matter (right panels).
Figure 3A Representative images of the vestibular nucleus (upper panels), cuneate nucleus (middle panels) and motor cortex layers II/III (lower panels) of SCA8 BAC mice at 2 months (left panels), 6 months (middle panels) and end-stage (10 months, right panels) stained with α-SerCT. Representative aggregates are indicated by black arrows. Corresponding quantifications of polySer aggregates for each region on the right (n=3; mean ± SEM; One-way ANOVA with Tukey's post-hoc test; * p<0.025, ** p<0.005, *** p<0.0005, **** p<0.0001)B Open field analysis of 2 month old animals show a significant decrease in ambulatory distance (cm) and ambulatory time (s) and a significant increase in resting time (s) (NT n=19, SCA8 BAC n=23, ** p<0.01, *** p<0.001; mean ± SEM; unpaired t test).
Figure 4A Disease-specific sensitivity to vacuolization after prolonged storage in ethanol shown by H&E in cerebellar white matter and brainstem of 10-12 month old end-stage SCA8 BAC mice but not in age matched NT (n=3) (panels 1&2). Demyelination shown by luxol fast blue staining (LFB) (panel 3) and axonal degeneration shown by α-SMI-32 (panel 4; degenerated axons are indicated by black arrows) observed in sites of polySer accumulation shown by α-SerCT (panel 5; representative aggregates are indicated by black arrows) in deep cerebellar white matter and brainstem in SCA8 BAC mice (lower panels) but not in NT mice (upper panels) (n=3).Demyelination shown by luxol fast blue staining (LFB) (panel 1) and axonal degeneration shown by α-SMI-32 (panel 2) observed sites of polySer accumulation shown by a-SerCT2 (panel 3) in deep cerebellar white matter was found in SCA8 autopsy tissue but not in control brains (n=3).C Immunohistochemistry using CC1 (α- APC) antibody shows significantly lower numbers of mature oligodendrocytes in SCA8 BAC mice compared to NT mice (NT n=5, SCA8 BAC n=5; **** p<0.0001; mean ± SEM; unpaired t test).D Immunofluorescence using α-GFAP antibody shows significant increase in astrogliosis in SCA8 BAC mice compared to NT mice (NT n=3, SCA8 BAC n=3, ** p<0.01; mean ± SEM; unpaired t test).E Cell death measured by lactase dehydrogenase (LDH) assay in T98 cells transfected with codon replacement constructs expressing polyGln (light blue) or polySer (dark blue). NT=nontransfected, EV=empty vector (n=5, ** p<0.003; *** p< 0.0003; mean ± SEM; unpaired t test).
Figure 5qRT-PCR showing Eif3f expression levels are increased 2-fold in SCA8 cerebellar white matter compared to SCA8 cerebellar grey matter (n=3, **p<0.01; mean ± SEM; unpaired t test).C Dot blot detection of polySer expressionin using α-FLAG antibody showing decrease in RAN polySer but not ATG-polySer when HEK293 cells are co-transfected with eIF3F siRNA. D Quantification of polySer detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test). E Detection of polyAla expression using α-HA antibody showing decrease in RAN polyAla but not ATG-polyAla when HEK293 cells are co-transfected with eIF3F siRNA. F Detection of polyGP expression using α-FLAG antibody showing a decrease in RAN polyGP when HEK293 cells are co-transfected with eIF3F siRNA.. G Quantification of polyAla and polyGP detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test).
Figure 1(B) Volcano plot of 8,701 mRNA isoforms from 2,931 genes with multiple isoforms. X-axis indicates a difference in percentage of each mRNA isoform within the gene (Δ%smg-2-N2). Y-axis indicates -log10 of p-value in Fisher's exact tests. Blue symbols indicate 219 mRNA isoforms significantly depleted from the smg-2 mutant (p<0.05, Δ%smg-2-N2<-10). Red symbols indicate 420 isoforms significantly enriched in the smg-2 mutant (p<0.05, Δ%smg-2-N2>10).(C, D) Length distribution of 3'UTRs (C) and proportions of putative PTC-containing isoforms (D) in 10,136 mRNA isoforms detected in N2 and/or smg-2 (All), the 219 isoforms depleted from and the 420 isoforms enriched in the smg-2 mutant.
Figure 2(B) Alternative splicing of sams-3, sams-4 and sams-5 in unfed and fed worms without or with a protein synthesis inhibitor emetine. Synchronized L1 larvae of an NMD-deficient strain KH1668: smg-2 (yb979) (lanes 1-3) and a wild-type strain N2 (lanes 4-6) were incubated in S-complete medium alone (lanes 1, 4), with a standard E. coli strain OP50 (OD600=10.0) (lanes 2, 5) or OP50 supplemented with 10 mg/ml emetine (lanes 3, 6) for 3 hours at 20°C. Total RNAs were extracted from whole animals and subjected to semi-quantitative RT-PCR, whose products were analyzed by capillary electrophoresis. Representative gel-like presentation is indicated (n=3). Schematic structure of each PCR product is indicated on the right. Open reading frames (ORFs) for full-length and truncated proteins are in orange and cyan, respectively. rpl-12 was used as an unaffected control.
Figure 3(A) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); sams-5 (gk147); sams-1 (ok2946) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 5), with OP50 (OD600=10.0) (lanes 2 and 6) and with OP50 supplemented with 25 mM L-Met (lanes 3 and 7) or 25 mM cycloleucine (cLeu) (lanes 4 and 8) for 3 hours at 20°C. The splicing patterns were analyzed and presented as in Figure 2B (n=3).(B, C) Western blot analysis of SAMS-1, SAMS-3 and SAMS-4 during larval development in the smg-2 (yb979) (B) and wild-type (C) backgrounds. Genotypes of the worms are smg-2 (w) and smg-2; sams-5; sams-1 (s) in (B) and wild-type (w) and sams-1 (s) in (C). Synchronized L1 larvae of each strain were incubated with OP50 at 20°C and subjected to Western blot analysis at indicated time points. Anti-β-tubulin (B) or coomassie brilliant blue (CBB) staining (C) was used as a loading control. Specificity of the antibodies is confirmed in Appendix Figure S12. Note that upregulation of SAMS-1 protein in the wild type during larval development in (C) is consistent with feeding-induced upregulation of sams-1 mRNA in Figure EV3D.
Figure 4(C) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); mett-10 (ok2204) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 3) or with OP50 (lanes 2 and 4) for 3 hours at 20°C and the splicing patterns were analyzed and presented as in Figure 2B.
Figure 5(A) Mass chromatograms of RNA fragments to detect m6A (top) or unmodified A (bottom) after in vitro incubation of 127-nt sams-3/sams-4 pre-mRNA (sequence available in Fig EV2) with recombinant full-length METT-10 protein in the presence (left) or absence (right) of 1 mM SAM. The sequence, m/z value, and charge state for each fragment are indicated on the right. Asterisks indicate non-specific signals.(B) The negatively-charged ions of RNase T1 fragment is decomposed in the instrument by collision-induced dissociation (CID) using helium gas. The product ions produced by CID are assigned on the sequence illustrated on the top right inset panel. Nomenclature of the product ions are described in the literature (McLuckey et al, 1992). Product ions of c and y series derive from the 5' and 3' termini of the fragment, respectively.
Figure 6A, B) m6A-IP specifically enriched sams mRNA isoforms that retain the AG dinucleotide at the distal/productive 3'SS. (A) Representative gel-like images (n=3) of semi-quantitative RT-PCR analysis of sams-3, sams-4 and sams-5 in input and immunoprecipitated (IP) RNAs from the smg-2 (yb979) mutant. The splicing patterns were analyzed and presented as in Figure 2B. (B) Relative enrichment of total and each of mRNA isoforms compared to eef-1A.1 mRNA by RT-qPCR. Error bars indicate standard error of mean (n=3). ( C) Examples of normalized Nanopore currents for unmodified and m6A-modified in-vitro transcribed sams-3/sams-4 RNAs at nucleotide positions -4 through +4 relevant to the m6A site. One hundred reads are lotted for each. Color codes are indicated. (D) Distribution of mean (top) and standard deviation (bottom) of normalized Nanopore currents at nucleotide positions -4 through +4 relevant to the m6A site for 5,000 of the unmodified and 2,680 of the m6A-modified sams-3/sams-4 RNAs and 85 of an endogenous sams-3 mRNA isoform E2/E3L. Color codes are indicated. Central bands represent the median, boxes represent the 25th and 75th percentiles, and whiskers represent the lowest and highest values. A red line at position 0 in the top panel indicates a cut-off line (mean current = 1.7557) that discriminates between the unmodified and m6A-modified sams-3/sams-4 RNAs with accuracies of 64.58% for both. (
Figure 1 (A RNA sequencing of CD31 and CD34 double-positive retinal endothelial cells from P5 wild-type mice. (A) RNA levels (FPKM; fragments per kilobase million) of marker genes of endothelial cells (green; CD31 (gene name PECAM1), CD34, von Willebrand Factor (vWF), tyrosine-protein kinase receptor Tie2 (TEK), VE-cadherin (CDH5), endoglin (ENG), CD146 (MCAM) and VEGFR2 (KDR)), astrocytes (blue, GFAP), immune cells (red; T-cells (CD3E), B-cells (CD19), all leucocytes (CD45, PTPRC), and monocytes/macrophages (F4/80, EMR1)), Müller glia cells (orange; aquaporin 4 (AQP4)), neurons (yellow; retinal ganglion cells (RNA binding fox-1 homolog 3, RBFOX3), amacrine cells (parvalbumin, PVALB), bipolar cells (PKC-α, PRKCA), horizontal cell (calbindin, CALB1), photoreceptors (rods, CD73 (NT5E); cones, transducing (GNAT1)), and retinal pigment epithelial cells (magenta; retinal pigment epithelium-specific 65 kDa protein (RPE65).  B) RNA sequencing of CD31 and CD34 double-positive retinal endothelial cells from P5 wild-type mice. (B) RNA levels of VASP (red), EVL (green) and Mena (blue) in the CD31 and CD34 double-positive P5 retinal endothelial cells. Error bars represent SEM; n = 18 animals (36 retinas) from six independent litters; four independent experiments. ***p<0.001, one-way ANOVA with Bonferroni's multi comparison test.  (C-E) Staining of Ena/VASP proteins in blood vessels of P5 mouse retinas. P5 wild-type mouse retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and VASP - (C), EVL- (D), or Mena-specific (E) antibodies (red). Yellow color in the merged images indicates the expression of VASP and EVL proteins in endothelial cells. No Mena protein expression was detected in P5 retinal endothelial cells (E). Representative images from three independent experiments are shown. Scale bars, 10 µm.
Figure 2 (A) Wild-type and EVL-/- P5 retinas were digested, labelled with CD31-, CD34- and EVL-specific antibodies and analyzed by flow cytometry. Endothelial cells were defined as CD31/CD34-double positive cells (gate indicated in the left panel) and analyzed for EVL expression (middle panel). Mean fluorescence intensity (MFI) of EVL in wild-type (magenta) and EVL-/- (black) cells is shown in the right panel. WT: n=3, EVL-/-: n=2; two retinas per animal. Error bars represent SEM.  (B-C) EVL protein expression in sparse/migrating wild-type (WT) and EVL-/- mouse brain endothelial cells (MBEC) and mouse lung endothelial cells (MLEC). Actin was used as loading control. Western blots are representative of three independent experiments.  (D) MLEC from wild-type (upper panel) and EVL-/- (lower panel) mice stimulated with 10 ng/ml VEGF were stained for actin (cyan) and EVL (magenta). Asterisks indicate focal adhesions, white arrows indicate filopodia and white arrowheads indicate the leading edge of lamellipodia. Representative images from four independent experiments are shown. Scale bar, 20 µm.