Journal: Nature Communications

Article Title: ELMO2 is an essential regulator of carotid artery development

doi: 10.1038/s41467-025-60105-9

Figure Lengend Snippet: a Representative images of control and Elmo2 −/− embryos at different developmental stages showing hemorrhages in the cervical region and subcutaneous edema (white arrowheads) in knockout mice. Scale bars, 2 mm. b Cross-section of an E12.5 Elmo2 +/− embryo stained with X-gal (blue) and Eosin (red) showing expression of Elmo2 in the cervical region. Higher magnification (bottom panel) shows relevant vascular structures: dorsal aorta (DA), third pharyngeal arch artery (3 rd PAA), jugular vein (JV), and jugular lymph sac (JLS). Scale bars, 2 mm. c Quantitation of area and perimeter of the 3 rd PAA in control and Elmo2 -/- E12.5 embryos. Mean ± SEM, n = 8. Welch’s t -test (area) and unpaired t -test (perimeter). d Quantitation of area and perimeter of the carotid artery (CA) in control and Elmo2 -/- E13.5 embryos. Mean ± SEM, n = 7. Welch’s t -test. e Confocal images of control and Elmo2 −/− E12.5 embryos stained for nuclei (DAPI, blue), veins and capillaries (EMCN, green), vascular smooth muscle cells (VSMCs, αSMA, red), lymphatic endothelial cell (EC) nuclei (Prox1, magenta) and ECs (CD31, gray). Higher magnification images correspond to the insets (outlined by white dashed line) in the respective overview images and highlight the 3 rd pharyngeal arch artery (PAA), jugular vein (JV), and jugular lymph sac (JLS). Scale bars, 200 µm (overview) and 50 µm (higher magnification). f Representative light sheet microscopy 3D images of control and Elmo2 −/− embryos at different developmental stages stained for ECs (CD31, red) and VSMCs (αSMA or SM22α, gray). Vascular structures of interest are indicated: pharyngeal arch arteries (PAA) or carotid arteries (CA). The schematic on the right shows the orientation of the embryos and the approximate area imaged in the respective developmental stages. Scale bars, 300 µm in each dimension.

Article Snippet: Silencer select siELMO2 (ThermoFisher, cat. #Hs00223006_m1), which covers most of the ELMO2 splice variants, was used for knockdown experiments by forward transfection.

Techniques: Control, Knock-Out, Staining, Expressing, Quantitation Assay, Microscopy

Journal: Nature Communications

Article Title: ELMO2 is an essential regulator of carotid artery development

doi: 10.1038/s41467-025-60105-9

Figure Lengend Snippet: a Confocal images of the 3 rd PAA from E12.5 embryos stained for endothelial cell (EC) junctions (VE-Cadherin, red) and arterial EC nuclei (SOX17, green). Scale bars, 10 µm. b EC nuclear area (left) and circularity (right) in the 3 rd PAA from E12.5 embryos. Mean ± SEM, n = 5 and Welch’s t -test (area), n = 4 and unpaired t -test (circularity). c Carotid artery (CA) cross-sections stained with the EC-luminal marker Podocalyxin (PODXL, gray) showing abluminal mislocalization (white arrowheads) in Elmo2 -/- E13.5 embryos. Scale bars, 25 µm. d 3 rd PAA stained for arterial EC nuclei (SOX17, green) and vascular smooth muscle cells (VSMCs, αSMA, gray) showing ectopic expression of αSMA in ECs (white arrowheads) from E12.5 Elmo2 −/− embryos. Scale bars, 10 µm. e Relative number of ERG + ECs with ectopic expression of αSMA normalized to vessel perimeter (PM) in the 3 rd PAA from E12.5 embryos. Mean ± SEM, n = 6. Unpaired t -test. f , g Confocal images ( f ) and corresponding quantitation ( g ) reflecting persistent expression of endomucin (EMCN, green) in arterial ECs (CD31, red) of E13.5 Elmo2 −/− CAs (white arrowheads). Scale bars, 25 µm. Mean ± SEM, n = 4. Welch’s t -test ( g ). h Super-resolution confocal images of the 3 rd PAA from E12.5 embryos stained for ECs (CD31, green) and VSMCs (αSMA, red). Scale bars, 10 µm. i , j Graphical representation ( i ) and corresponding quantitative analysis ( j ) of VSMC-actin bundles’ (αSMA, blue) alignment with respect to ECs’ longest axis (CD31, orange) in E12.5 embryos. Data in ( j ) represented as Mean ± SEM, n = 6. Unpaired t -test. k Confocal images of 3 rd PAA cross-sections stained for VSMC markers (αSMA, red or SM22α, gray) and color-coded representation of signal intensity in E12.5 embryos. Panel on the right shows merged channels including staining for nuclei (DAPI, blue) and ECs (CD31, green). Scale bars, 10 µm. l Intensity profile of αSMA and SM22α immunosignals from 3 rd PAA cross-sections of E12.5 embryos. The green dashed line indicates the position of ECs (ectopic expression of αSMA and SM22α in Elmo2 -/- ). The black dashed line indicates the position of the first VSMCs.

Article Snippet: Silencer select siELMO2 (ThermoFisher, cat. #Hs00223006_m1), which covers most of the ELMO2 splice variants, was used for knockdown experiments by forward transfection.

Techniques: Staining, Marker, Expressing, Quantitation Assay

Journal: Nature Communications

Article Title: ELMO2 is an essential regulator of carotid artery development

doi: 10.1038/s41467-025-60105-9

Figure Lengend Snippet: a Uniform manifold approximation and projection (UMAP) plot of different cell types in the cervical region of E12.5 mouse embryos. b Violin plot showing average expression level of Elmo2 transcript in cells from the different samples analyzed. c Aggregate heatmaps showing the fraction of cells expressing Elmo2 and the mean expression of Elmo2 in the different cell types identified. Color represents a scaled fraction or expression level. d UMAP plot of the endothelial cell population in the cervical region of E12.5 mouse embryos. Colors represent different cell subclusters. e UMAP plot of the endothelial cell population in the cervical region of E12.5 mouse embryos. Colors represent different samples ( Elmo2 +/+ , blue; Elmo2 −/− , orange). The red arrowhead indicates a group of cells within the arterial population which is only present in the knockout embryos. f Volcano plot of differentially expressed genes between endothelial cells from control and Elmo2 −/− E12.5 embryos. Blue dots, p -adjusted value < 1 −10 ; orange dots, p -adjusted value < 1 −10 and log 2 fold change > 2.0 or < − 2.0. Wald test and multiple testing correction by independent hypothesis weighting (IHW). g UMAP plots depicting the expression pattern of selected genes, which appear upregulated in endothelial cells from Elmo2 −/− . Color represents the scaled expression level. h UMAP plot of the different cellular subclusters identified within the mesenchymal stromal cell (MSC) population. i Color-coded UMAP plot of the MSC cluster comparing control ( Elmo2 +/+ , blue) and knockout ( Elmo2 −/− , orange) cell populations. j Volcano plot of differentially expressed genes between MSCs from control and Elmo2 −/− E12.5 embryos. Blue dots, p -adjusted value < 1 −10 ; orange dots, p -adjusted value < 1 −10 and log 2 fold change > 2.0 or < − 2.0. Wald test and multiple testing correction by independent hypothesis weighting (IHW). k Volcano plots showing differentially expressed genes in the indicated cell populations after comparison of control ( Elmo2 +/+ ) and Elmo2 −/− E12.5 samples. Blue dots, p -adjusted value < 1 −10 ; orange dots, p -adjusted value < 1 −10 and log 2 fold change > 2.0 or < − 2.0. Wald test and multiple testing correction by independent hypothesis weighting (IHW).

Article Snippet: Silencer select siELMO2 (ThermoFisher, cat. #Hs00223006_m1), which covers most of the ELMO2 splice variants, was used for knockdown experiments by forward transfection.

Techniques: Expressing, Knock-Out, Control, Comparison

Journal: Nature Communications

Article Title: ELMO2 is an essential regulator of carotid artery development

doi: 10.1038/s41467-025-60105-9

Figure Lengend Snippet: a Representative images of control and Elmo2 ΔSMC E13.5 embryos showing no macroscopic defects upon Elmo2 deletion in vascular smooth muscle cells (VSMCs). Scale bars, 2 mm. b Confocal overview images of transverse sections from control and Elmo2 ΔSMC embryos stained for nuclei (DAPI, blue), endothelial cells (ECs, CD31, red), and VSMCs (SM22α, gray) showing mild dilation of the carotid artery (CA) in E13.5 and E15.5 mutant embryos. Scale bars, 200 µm. c Quantitation of area and perimeter of the carotid arteries in E13.5 and E15.5 Elmo2 ΔSMC embryos normalized to control (set as 1). Mean ± SEM, n = 6. Welch’s t -test (area E13.5) and unpaired t -test (area E15.5 and perimeter at all stages). d Confocal images of carotid arteries (CA) from E13.5 control and Elmo2 ΔSMC embryos stained for nuclei (DAPI, blue), ECs (CD31, green), veins/capillaries (EMCN, magenta), and VSMCs (αSMA, red; SM22α, gray). Scale bars, 25 µm. e Recombination analysis in Tagln-Cre;R26-mTmG E13.5 embryos. Representative confocal images of the carotid artery (CA, white dashed line square in overview image) stained for nuclei (DAPI, blue), recombined cells (GFP, green), ECs (CD31, red), and VSMCs (SM22α, gray). Higher magnification images show GFP expression restricted to VSMCs and surrounding mesenchymal cells. Scale bars, 200 µm (overview) and 20 µm (higher magnification).

Article Snippet: Silencer select siELMO2 (ThermoFisher, cat. #Hs00223006_m1), which covers most of the ELMO2 splice variants, was used for knockdown experiments by forward transfection.

Techniques: Control, Staining, Mutagenesis, Quantitation Assay, Expressing

Journal: Nature Communications

Article Title: ELMO2 is an essential regulator of carotid artery development

doi: 10.1038/s41467-025-60105-9

Figure Lengend Snippet: a Recombination analysis of Wnt1-Cre2;R26-mTmG E13.5 embryos. Representative confocal images of the carotid artery (CA, white dashed line square in overview image) stained for nuclei (DAPI, blue), recombined cells (GFP, green), endothelial cells (ECs, CD31, red), and vascular smooth muscle cells (VSMCs, SM22α, gray). Scale bars, 200 µm (overview) and 25 µm (higher magnification). b Representative images of control and Elmo2 ΔNCC E13.5 embryos showing hemorrhages in the cervical region and subcutaneous edema (white arrowheads) in the neural crest-specific knockout mice. Scale bars, 2 mm. c Confocal overview images of cross-sections from control and Elmo2 ΔNCC E13.5 embryos stained for nuclei (DAPI, blue), recombined cells (GFP, green), ECs (CD31, red), and VSMCs (SM22α, gray) showing aneurysm formation in the carotid artery (CA) of neural crest-specific knockout mice. Scale bars, 500 µm. d Quantitation of area and perimeter of the carotid arteries in control and Elmo2 ΔNCC E13.5 embryos. Mean ± SEM, n = 5. Mann-Whitney test. e Confocal images of the carotid artery from control and Elmo2 ΔNCC E13.5 embryos stained for VSMCs (SM22α, gray; αSMA, red) and color-coded representation of signal intensity (right panels). Scale bar, 10 µm. f High-resolution confocal images of carotid arteries stained for ECs (CD31, green), VSMCs (αSMA, red), and nuclei (DAPI, blue) showing aberrant alignment of αSMA-bundles with respect to the longest axis of ECs in E13.5 Elmo2 ΔNCC embryos. Scale bars, 10 µm. g Quantitation of average relative angle between the ECs’ longest axis and VSMC-actin bundles in control and Elmo2 ΔNCC E13.5 embryos. Mean ± SEM, n = 4. Welch’s t -test. h Confocal images of carotid arteries (CA) stained for veins/capillaries (EMCN, green) and ECs (CD31, red) showing persistent expression of endomucin in the CA (white arrowheads) of E13.5 Elmo2 ΔNCC embryos. Scale bars, 100 µm. i Confocal images of carotid arteries stained for nuclei (DAPI, blue), ECs (CD31, red), recombined cells (GFP, green) and VSMCs (SM22α, gray) showing abnormal expression of SM22α in ECs (white arrowheads) and discontinuities in the endothelial lining (yellow arrowhead) of E13.5 Elmo2 ΔNCC embryos. Scale bars, 25 µm.

Article Snippet: Silencer select siELMO2 (ThermoFisher, cat. #Hs00223006_m1), which covers most of the ELMO2 splice variants, was used for knockdown experiments by forward transfection.

Techniques: Staining, Control, Knock-Out, Quantitation Assay, MANN-WHITNEY, Expressing

Journal: Nature Communications

Article Title: ELMO2 is an essential regulator of carotid artery development

doi: 10.1038/s41467-025-60105-9

Figure Lengend Snippet: a ELMO2 expression (RT-qPCR) in siControl and siELMO2 HBVSMCs at different timepoints (hours, h). Mean ± SEM, n = 3. Welch’s t -test. b ELMO2 immunoblot from siControl and siELMO2 HBVSMCs at different timepoints. Molecular weight markers (kDa) are indicated. c , d Area covered by attached HBVSMCs at different timepoints ( c ) and representative images ( d ) of siControl (green) and siELMO2 (red) cells stained for nuclei (DAPI, blue) and actin (Phalloidin, gray). Mean ± SEM, n = 3. Welch’s t -test (30 min) and unpaired t test (all other time points). Scale bars, 50 µm. e High magnification images of siControl and siELMO2 HBVSMCs stained for nuclei (DAPI, blue) and actin (Phalloidin, gray) reveal membrane blebbing upon ELMO2-knockdown 10 min after seeding. Scale bars, 25 µm. f , g Representative images ( f ) and average cell area ( g ) of siControl (green) and siELMO2 (red) HBVSMCs growing in a 3D fibrin gel. Scale bars, 50 µm. Mean ± SEM, n = 3. Unpaired t -test. h , i Representative images ( h ) and contractility analysis ( i ) of siControl (green) and siELMO2 (red) HBVSMCs upon carbachol-treatment. Scale bars, 25 µm. Mean ± SEM, n = 3. Welch’s t -test. j , k Representative images ( j ) and contractility analysis ( k ) of siControl and siELMO2 HBVSMCs 12 h after seeding in collagen matrix. Scale bars, 5 mm. Mean ± SEM, n = 4. Unpaired t -test. l , m Immunoblot ( l ) and corresponding quantitation ( m ) of G-actin:F-actin ratio in siControl and siELMO2 HBVSMCs. Molecular weight marker (kDa) is indicated. Mean ± SEM, n = 3. Unpaired t test. n Differences in active RAC1 in siControl and siELMO2 -treated HBVSMCs. Mean ± SEM, n = 3. Unpaired t -test. o Representative images of siControl and siELMO2 HBVSMCs treated with jasplakinolide (JAS) and stained for nuclei (DAPI, blue) and actin (Phalloidin, gray). Note rescued filopodia formation during cell spreading 10 min after seeding. Scale bars, 25 µm. p Area covered by attached siControl and siELMO2 HBVSMCs after vehicle or jasplakinolide (JAS) treatment. Mean ± SEM, n = 3. Ordinary one-way ANOVA. q , r Collagen gel contraction assay ( q ) and corresponding quantitation ( r ) showing rescued contractility in jasplakinolide (JAS)-treated siELMO2 HBVSMCs. Scale bars, 5 mm. Mean ± SEM, n = 3. Ordinary one-way ANOVA.

Article Snippet: Silencer select siELMO2 (ThermoFisher, cat. #Hs00223006_m1), which covers most of the ELMO2 splice variants, was used for knockdown experiments by forward transfection.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Molecular Weight, Staining, Membrane, Knockdown, Quantitation Assay, Marker, Collagen Gel Contraction Assay

Journal: Nature Communications

Article Title: ELMO2 is an essential regulator of carotid artery development

doi: 10.1038/s41467-025-60105-9

Figure Lengend Snippet: a Scheme of 4-hydroxytamoxifen (4-OHT) injection (red arrowheads) and sample collection (analysis, black arrowhead) timepoints. b Representative overview confocal images from cross-sections sections of control and Elmo2 iko E13.5 embryos stained for nuclei (DAPI, blue), recombined cells (GFP, green), endothelial cells (ECs, CD31, red) and vascular smooth muscle cells (VSMCs, SM22α, gray) showing aneurysm formation and merging of carotid arteries (CA) in mutant embryos. Scale bars, 200 µm. c Quantitation of area and perimeter of CAs from control and Elmo2 iko E13.5 embryos after 4-OHT injection at E8.5-E9.5. Mean ± SEM, n = 6. Mann-Whitney test. d Immunoblot of whole-tissue lysates from control and Elmo2 iko E13.5 embryos after 4-OHT injection at E8.5-E9.5. Molecular weight marker (kDa) is indicated. e Scheme of 4-OHT injection (red arrowheads) and sample collection (analysis, black arrowhead) timepoints. f Representative overview confocal images from cross-sections of control and Elmo2 iko E13.5 embryos stained for nuclei (DAPI, blue), recombined cells (GFP, green), ECs, (CD31, red), and VSMCs (SM22α, gray) showing mild dilation of carotid arteries (CA) in mutant embryos. Scale bars, 200 µm. g Quantitation of area and perimeter of CAs from control and Elmo2 iko E13.5 embryos after 4-OHT injection at E10.5-E11.5. Mean ± SEM, n = 5. Welch’s t -test. h Immunoblot of whole-tissue lysates from control and Elmo2 iko E13.5 embryos after 4-OHT injection at E10.5-E11.5. Molecular weight marker (kDa) is indicated. i Scheme of 4-OHT injection (red arrowheads) and sample collection (analysis, black arrowhead) timepoints. j Representative overview confocal images from cross-sections of control and Elmo2 iko E15.5 embryos stained for nuclei (DAPI, blue), recombined cells (GFP, green), ECs, (CD31, red) and VSMCs (SM22α, gray) showing no obvious phenotypic changes in the carotid arteries (CA) of mutant embryos. Scale bars, 200 µm. k Quantitation of area and perimeter of CAs from control and Elmo2 iko E15.5 embryos after 4-OHT injection at E10.5-E11.5. Mean ± SEM, n = 4. Welch’s t -test. l Immunoblot of whole-tissue lysates from control and Elmo2 iko E15.5 embryos after 4-OHT injection at E10.5-E11.5. Molecular weight marker (kDa) is indicated.

Article Snippet: Silencer select siELMO2 (ThermoFisher, cat. #Hs00223006_m1), which covers most of the ELMO2 splice variants, was used for knockdown experiments by forward transfection.

Techniques: Injection, Control, Staining, Mutagenesis, Quantitation Assay, MANN-WHITNEY, Western Blot, Molecular Weight, Marker

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of Ephexin4 at Ser-41 contributes to chromosome alignment via RhoG activation in cell division

doi: 10.1016/j.jbc.2024.108084

Figure Lengend Snippet: Active RhoG-G12V is localized to the plasma membrane. A , expression of WT and mutant Ephexin4 were induced by treating HeLa S3/WT, HeLa S3/S41A, and HeLa S3/S41E cells with Dox, and their cell lysates were incubated with glutathione beads precoated with the N-terminal region of ELMO2. The pull down samples were analyzed by Western blot analysis. B , HeLa S3/RhoG WT and HeLa S3/RhoG G12V cells were transfected with Ephexin4-targeting (E, #3, 250 nM) or nontargeting (C, 250 nM) siRNA. After 7.5 h of transfection, the cells were treated with 4 mM thymidine with or without Dox (WT, 0.5 μg/ml; G12V, 0, 0.1, 0.3, 0.5, 0.7 μg/ml) for 24 h. After a 6-h release from thymidine, the cells were treated with 6 μM RO-3306 for 10.5 h, followed by release from RO-3306 for 30 min. The cells were then treated with 40 μM MG132 for 90 min, and whole-cell lysates of M-phase cells collected by mitotic shake-off were subjected to Western blot analysis. C – F , HeLa S3/RhoG WT and HeLa S3/RhoG G12V cells were transfected with Ephexin4-targeting (E, #3, 250 nM) or nontargeting (C, 250 nM) siRNA. After 7.5 h of transfection, the cells were treated with or without 0.5 μg/ml Dox for 20.5 h. Then, the cells were treated with 6 μM RO-3306 and continuously with Dox for 20 h. After a 30-min release from RO-3306, the cells were treated with 40 μM MG132 for 90 min and then fixed for DNA and RhoG staining. The fluorescence intensity of RhoG at the plasma membrane was quantified in metaphase cells and is plotted as the mean ± SD of three ( C ) or two ( E ) independent experiments ( B , n ≥ 34 cells; D , n ≥ 35 cells per condition). Dunnett test was used to determine p -values. Scale bars represent 10 μm ( C , E ). The percentage of cells exhibiting chromosome misalignment is plotted as the mean ± SD of three independent experiments ( D , n ≥ 172 cells; F , n ≥ 169 cells per condition). Tukey’s test was used to determine p -values ( D , F ).

Article Snippet: To construct GST-ELMO2, the RhoG-binding domain–included N-terminal region (aa 1–362) of ELMO2 cDNA (GenScript Japan, Clone ID, OHu02300C) was amplified by PCR using the primer set, 5′-CTCGGATCCGAATTCATGCC-3′ and 5′-TTTAAGCTTCTACATGGCTGGATTGATGTGGTTGG-3′, generating DNA fragment harboring BamHI and HindIII sites.

Techniques: Clinical Proteomics, Membrane, Expressing, Mutagenesis, Incubation, Western Blot, Transfection, Staining, Fluorescence