Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: Five compounds found to induce increased proliferation on the primary screen of Megf10 shRNA C2C12 myoblasts, with molecular targets in mammalian, zebrafish and Drosophila noted

Article Snippet: Immortalized myoblast models generated by Megf10 shRNA knockdown C2C12 myoblasts (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) with 20% fetal bovine serum (Sigma), penicillin (50 units/mL) and streptomycin (50 μg/mL; Invitrogen).

Techniques: shRNA

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: In vitro secondary drug screens conducted on C2C12 myoblasts and primary mouse myoblasts. (A) Proliferation assay performed with the five candidate compounds on C2C12 cells transfected with V5-tagged C774R mutant Megf10. Drug compounds were administered at 24 h of culture, and CyQUANT assays were performed at 48 h of culture. Statistically significant treatment effects are seen for sertraline (Sert) and escitalopram (Esci). The vehicle was DMSO (Veh). Horizontal bars represent the mean ± S.E.M. from 12 wells in a 96-well plate. On ANOVA, P < 0.001. On Bonferroni post hoc t-test, **P < 0.01; ***P < 0.001. (B) Proliferation assay performed with the five candidate compounds on primary myoblast cultures from wild type and Megf10−/− mice. Drug compounds were administered at 24 h of culture, and CyQUANT assays were performed at 48 h of culture. Statistically significant treatment effects are seen for sertraline and escitalopram. Scatterplots are presented, with horizontal bars representing the mean ± S.E.M. from 8 wells in a 96-well plate. On two-way ANOVA, P < 0.05. On Sidak's multiple comparisons test, *P < 0.05. (C) On migration assay, all five drug candidates yielded improved migration at 24 and 72 h, but the effect was more pronounced for sertraline and escitalopram at 72 h. Irregular yellow lines show borders between cellular and acellular zones for each image. Scale bar, 5 mm. (D) Quantifications of residual clear areas on the migration assay using ImageJ software at indicated time points. On two-way ANOVA, P < 0.05. On Sidak's multiple comparisons test *P < 0.05; **P < 0.01; n = 5 images. (E) An adhesion assay was performed on Megf10-deficient myoblasts and scrambled shRNA control myoblasts using the five candidate compounds. Megf10 shRNA-treated cells recovered significantly with Sert, Esci and Losa compounds at 60 min time points. Horizontal bars represent the mean fluorescence intensities of the adherent cells ± S.E.M. from two independent experiments, n = 2 per experiment. On two-way ANOVA, P < 0.05. On Sidak's multiple comparisons test, *P < 0.05.

Article Snippet: Immortalized myoblast models generated by Megf10 shRNA knockdown C2C12 myoblasts (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) with 20% fetal bovine serum (Sigma), penicillin (50 units/mL) and streptomycin (50 μg/mL; Invitrogen).

Techniques: In Vitro, Proliferation Assay, Transfection, Mutagenesis, CyQUANT Assay, Migration, Software, Cell Adhesion Assay, shRNA, Control, Fluorescence

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: Megf10-deficient zebrafish. Using brightfield microscopy, (A) the wild-type zebrafish maintains a straight posture, while (B) the megf10 mutant displays significant tail bending (a manifestation of severe dorsal muscle weakness). Birefringence microscopy, (C) the wild type zebrafish refracts polarized light off its dorsal muscles, whereas (D) the megf10-deficient zebrafish has black patches indicating muscle wasting, disorganized muscle fibers and myofiber degeneration pathology. Zebrafish are photographed at 4 dpf. Scale bar, 500μm. Preliminary treatment studies of sertraline and escitalopram in megf10-deficient zebrafish. Each drug cohort included 25 fish from heterozygous matings, and all fish were analyzed at 5 dpf. (E) Vehicle treated fish showed significant tail bending pathologies that were prevented with (F) 1 μm sertraline and with (G) 1 μm escitalopram. (H) Survival curves show markedly improved survival for all treatment groups. Percent of living fish were determined by detecting heartbeats. Data represent mean ± SEM relative to controls. On two-way ANOVA, P < 0.05. Sidak's multiple comparisons test results are indicated on the graph; *P < 0.05, **P < 0.01. Scale bar, 1 mm.

Article Snippet: Immortalized myoblast models generated by Megf10 shRNA knockdown C2C12 myoblasts (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) with 20% fetal bovine serum (Sigma), penicillin (50 units/mL) and streptomycin (50 μg/mL; Invitrogen).

Techniques: Microscopy, Mutagenesis, Muscles

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: Summary of three families enrolled with MEGF10 myopathy

Article Snippet: Immortalized myoblast models generated by Megf10 shRNA knockdown C2C12 myoblasts (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) with 20% fetal bovine serum (Sigma), penicillin (50 units/mL) and streptomycin (50 μg/mL; Invitrogen).

Techniques: Mutagenesis

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: Analysis of sertraline treatment in human iPSC-derived MEGF10-deficient myoblasts and murine Megf10-deficient myoblasts. (A) iPSC-derived human myoblasts representing MEGF10 deficiency (02-1, 03-1, 27-1 27-3, 27-4) showed impaired proliferation compared to asymptomatic carrier parents (02-2, 02-3) and a healthy control (H) at 48 and 72 h time points. A two-way ANOVA showed P < 0.0001. Bonferroni post test results are indicated on the graph: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (B) Human MEGF10-deficient myoblasts showed significantly improved proliferation compared to untreated myoblasts at 72 h after 1 μm sertraline hydrochloride treatment. Two-way ANOVA showed P < 0.0001. Bonferroni post-test results are depicted on the graph: *P < 0.05; **P < 0.01; ****P < 0.0001. (C) A migration assay performed on iPSC-derived human myoblasts demonstrated marked impairment in migration for MEGF10-deficient myoblasts compared to carriers and a healthy control. The yellow lines mark the borders between cellular and acellular zones. Sertraline treatment induced a marked improvement in migration patterns compared to untreated controls at 72 and 120 h. Scale bar, 5mm; +, sertraline treatment; −, no drug treatment. (D,E) Quantification of clear areas on migration assay using ImageJ software at indicated time points. On two-way ANOVA, P < 0.05. Sidak's multiple comparisons test *P < 0.05, **P < 0.01. (F) Expression levels of MEGF10, NOTCH1, HEY1, PAX7, ITGA7 and MYOD are compared via RT-PCR in MEGF10-deficient iPSC-derived myoblasts (02-1) versus carrier parent myoblasts (02-3). Data represent mean ± SEM relative to controls performed in triplicate and are presented as percentages of control myoblast expression levels. On two-way ANOVA, P < 0.05. Sidak's multiple comparisons test results are indicated on the graph; *P < 0.05, **P < 0.01; ***P < 0.001. (G) Notch1 protein expression is increased in Megf10 shRNA knockdown and wild-type C2C12 myoblasts after sertraline hydrochloride treatment, most notably at 24 and 48 h. Notch1 augmentation attenuates in the Megf10 shRNA myoblasts by 72 h. Gapdh was used as a loading control. Immunoblot images (top) and their quantification by ImageJ (bottom) are shown. The ImageJ graphs represent data from four independent experiments. Two-way ANOVA shows P < 0.001. Tukey's multiple comparison post-test results are depicted on the graph; *P < 0.05.

Article Snippet: Immortalized myoblast models generated by Megf10 shRNA knockdown C2C12 myoblasts (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) with 20% fetal bovine serum (Sigma), penicillin (50 units/mL) and streptomycin (50 μg/mL; Invitrogen).

Techniques: Derivative Assay, Control, Migration, Software, Expressing, Reverse Transcription Polymerase Chain Reaction, shRNA, Knockdown, Western Blot, Comparison

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: Sertraline treatment reactivates the Notch pathway in murine Megf10-deficient myoblasts and human MEGF10-deficient myoblasts. (A) Cell proliferation assays were performed on Megf10-deficient and wild type C2C12 myoblasts that were treated with sertraline alone, sertraline with DAPT and sertraline with serotonin. Three individual experiments with 16 wells are shown. A two-way ANOVA test was performed on different groups and conditions with Sidak’s post test, scatter plots representing the mean absorbance ± S.E.M. (****P < .0001). (B) Cell proliferation assays were performed on MEGF10-deficient human myoblasts (02-1, 03-1, 27-1) and healthy control myoblasts (H). The scatter plots indicate two independent experiments. A two-way ANOVA test was performed on different groups and conditions with Sidak’s post test, scatter plots representing the mean absorbance ± S.E.M. (C) The heat map represents mRNA expression levels measured via RT-PCR of human Notch signaling genes in human MEGF10-deficient myoblasts treated with sertraline compared to untreated healthy control myoblasts. Genes are located horizontally and samples vertically in the order given. Each sample is represented by the mean of the gene expression value of the samples within that subgroup. Healthy control is the calibrator. Blue indicates down regulation. (D) The heat map represents mRNA expression levels measured via RT-PCR of human Notch signaling genes in human MEGF10-deficient myoblasts treated with sertraline compared to untreated healthy control myoblasts. No drug treatment is the calibrator. Yellow indicates relative upregulation, and blue indicates relative downregulation. (E) Significant fold changes for the indicated genes were plotted as the mean fold change relative to the expression of the same genes in healthy untreated control subjects. Multiple unpaired t-tests were performed and P < 0.05 was considered significant.

Article Snippet: Immortalized myoblast models generated by Megf10 shRNA knockdown C2C12 myoblasts (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) with 20% fetal bovine serum (Sigma), penicillin (50 units/mL) and streptomycin (50 μg/mL; Invitrogen).

Techniques: Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal:

Article Title: MEGF10 Association with Schizophrenia

doi: 10.1016/j.biopsych.2007.11.003

Figure Lengend Snippet: The MEGF10 gene and the LD and haplotype structure of the samples used in this study. A. Gene structure and the relative position of the typed SNPs. Rs27652 was too close to rs27388, it was not shown in the picture. In the figure, exons were illustrated by the long horizontal bars. The 3′ and 5′ UTRs were illustrated by the short bars. B. LD and haplotype structures of the screening subsample. LDs shown were D’. Haplotypes were labeled by letters on the left and their frequencies were listed on the right. Risk haplotype was highlighted. C. LD and haplotype structures of the replication subsample. D. LD and haplotype structures of the ICCSS sample.

Article Snippet: Real-time quantitative PCRs were conducted with TaqMan expression probe (Hs01002798_m1) for the MEGF10 gene, and human TATA box binding protein ( TBP ) gene was used as internal reference.

Techniques: Labeling

Journal:

Article Title: MEGF10 Association with Schizophrenia

doi: 10.1016/j.biopsych.2007.11.003

Figure Lengend Snippet: ANCOVA analyses of the MEGF10 gene expressions in the Stanley subjects. The expression level was the ΔCT between MEGF10 and the house keeping gene TBP. The error bars were standard errors. A. Comparison of expressions between the control and schizophrenia subjects. B. Comparison of expressions between the control and schizophrenia subjects with the inclusion of the genotypes of rs27388.

Article Snippet: Real-time quantitative PCRs were conducted with TaqMan expression probe (Hs01002798_m1) for the MEGF10 gene, and human TATA box binding protein ( TBP ) gene was used as internal reference.

Techniques: Expressing, Comparison, Control

Journal: Cell reports

Article Title: Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells.

doi: 10.1016/j.celrep.2023.113137

Figure Lengend Snippet: Figure 1. Establishment and validation of MEGF11-KD in GCs (A) Schematic of domain structures of MEGF11. (B) Positions and sequences of the three candi- date MEGF11 shRNAs. (C) Images of HEK 293T cells transfected with pEGFP or pEGFP-MEGF11-UTR together with shRNA expression plasmids. (D) Diagram of AAV-SicoR-shRNA (top), and a representative image of dT expression in an ICR mouse subjected to AAV-SicoR-shScr injection (bottom). (E and F) qPCR results of Megf11, Megf10, and Megf12 mRNA in the cerebellum of non-injected control (CTR) mice (n = 12), or mice expressing shScr (shS, n = 12), type 1 (T1, n = 14), type 2 (T2, n = 12), or type 3 (T3, n = 9) MEGF11 shRNA, obtained using TaqMan probes detecting exons 2 and 3 regions (E) or a primer pair detecting 30 UTR (F). Results of Megf11 in (E) are also shown in (F) for direct comparison. (G) Schematic diagram showing the injection of AAV-SicoR-shRNA into lobule IV/V of PV-Cre mice (left), and a representative image of resultant dT expression (right). (H) Experimental design to test Megf11 mRNA expression in PCs and MLIs, or in GCs by the TRAP technique. (I) qPCR results of Megf11 mRNA purified from PCs and MLIs (n = 5 for shScr, n = 6 for shMegf11), or from GCs (n = 7 for shScr, n = 8 for shMegf11), of PV-Cre mouse cerebella with shScr or shMegf11 expression. Data were normalized to those of CTR mice (E and F) or of mice expressing shScr (I). Error bars in this and subsequent figures indicate SEM. The roman numerals in the images of this and subse- quent figures indicate the cerebellar lobules. ***p < 0.001, **p < 0.01, *p < 0.05, one-way ANOVA followed by the Bonferroni test (E and F) or Stu- dent’s t test (I). The exact p values for the datasets in this and subsequent figures are shown in Table S1.

Article Snippet: TaqMan probes used for Megf11, Megf10, Megf12, and Gapdh were Mm00553032_m1, Mm01257625_m1, Mm00546891_m1, and Mm99999915_g1 (Thermo Fisher Scientific), respectively.

Techniques: Biomarker Discovery, Transfection, shRNA, Expressing, Injection, Control, Comparison

Journal: The Journal of Neuroscience

Article Title: Megf10 Is a Receptor for C1Q That Mediates Clearance of Apoptotic Cells by Astrocytes

doi: 10.1523/jneurosci.3850-15.2016

Figure Lengend Snippet: Figure 1. Megf10 is necessary for apoptotic cell uptake by astrocytes, and its deficiency results in accumulation of apoptotic cells in the developing CB. A, Megf10 mRNA expression in different organsfromC57B6WTmalemiceasmeasuredbyRT-PCR(n3).B,Megf10expressionintheCBofP7WT,Megf10/,andMegf10/mice(n3–5;p0.01).C,Quantificationofanti-CC3 staining in the CB of WT, Megf10/, and Megf10/ P7 mice showing that Megf10/ and Megf10/ mice had a significant accumulation of apoptotic cells (Figure legend continues.)

Article Snippet: A full-length human Megf10 (hMegf10) cDNA clone (catalog #TC104847; Origene Technologies) was subcloned into the pCDNA3.1 vector (catalog #V790-20; Invitrogen Life Technologies).

Techniques: Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: Megf10 Is a Receptor for C1Q That Mediates Clearance of Apoptotic Cells by Astrocytes

doi: 10.1523/jneurosci.3850-15.2016

Figure Lengend Snippet: Figure2. Megf10 does not bind directly PS but binds C1q with high affinity. A, Schematic depiction of secreted ex-hMegf10 tagged with His and Flag tag. Western blot confirmed that the secreted and purified protein is a single 100 kDa band as predicted. B, Dot blots showing binding of ex-hMegf10 to various membrane lipids and no binding to PS. C, Quantitative densitometry of the blot presented in B (***p 0.0001; n 3 from 3 independent experiments). D, Representative dot blot of binding of ex-hMegf10 to immobilized C1q and quantitativedensitometryshowingasignificantincreaseinbindingcomparedwithsecondaryantibodyalone(*p0.05;n4).E–G,Label-freesurfaceplasmonresonanceconfirming that there is no binding to PS (F) and showing affinity of ex-Megf10 to C1q in the nanomolar range with a highly significant ( 2 1) as measured by full kinetic analysis (G). H, HEK expressingcontrolGFPorfull-lengthhMegf10–GFP(FLM10-GFP)wereincubatedwiththeindicatedamountsofAlexa-Fluor647–C1q(C1q-647)for1handmeasuredbyflowcytometry. FL M10 analysis was done on the GFP-positive transfected population (FL M10-GFP) and the GFP-negative nontransfected population (FL M10-GFP; n 3). I, J, HEK cells expressing unlabeled hMegf10 were incubated with C1q-647 (red) (I) or C1q-647 (red) and apoptotic cells (white) (J), washed, and stained with rabbit anti-Megf10 antibody (green) and DAPI nuclear stain (blue). HEK–Megf10 cells take up C1q-647 (I) and apoptotic cells coated with C1q-647 (J). Scale bar, 20 m.

Article Snippet: A full-length human Megf10 (hMegf10) cDNA clone (catalog #TC104847; Origene Technologies) was subcloned into the pCDNA3.1 vector (catalog #V790-20; Invitrogen Life Technologies).

Techniques: FLAG-tag, Western Blot, Purification, Binding Assay, Membrane, Dot Blot, Transfection, Expressing, Incubation, Staining

Journal: The Journal of Neuroscience

Article Title: Megf10 Is a Receptor for C1Q That Mediates Clearance of Apoptotic Cells by Astrocytes

doi: 10.1523/jneurosci.3850-15.2016

Figure Lengend Snippet: Figure3. EMARDDmutationsimpairengulfmentofapoptoticcells.A–D,Anti-Megf10stainingofHEKcellsexpressingcontrol(A),full-lengthhMegf10(B),hMegf10–C326R(C),andhMegf10– C774R(D)validatingexpressionofMegf10onthecellsurface.E,Meanfluorescentintensity(MFI)ofGFP-positivecellsconfirmsimilarexpressionlevelsofMegf10inalltransfectedcells.F,Analysis of UV MEF uptake by HEK transfected cells shows a defect in engulfment in hMegf10 mutations C326R (22% reduction) and C774R (53% reduction) compared with full-length hMegf10 (***p 0.0001;n9from3independentexperimentrunintriplicates).G,H,TransfectedHEKcellswereincubatedwiththeindicatedamountsofAlexa-Fluor647–C1qfor1h.Percentageofuptake(H) and MFI (I) was measured by flow cytometry.

Article Snippet: A full-length human Megf10 (hMegf10) cDNA clone (catalog #TC104847; Origene Technologies) was subcloned into the pCDNA3.1 vector (catalog #V790-20; Invitrogen Life Technologies).

Techniques: Transfection, Flow Cytometry

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: Five compounds found to induce increased proliferation on the primary screen of Megf10 shRNA C2C12 myoblasts, with molecular targets in mammalian, zebrafish and Drosophila noted

Article Snippet: Megf10 mutant zebrafish (megf10 sa13029 ) contain a T to A nonsense mutation in exon 5 of zebrafish megf10 and were obtained from the Zebrafish International Resource Center (Eugene, OR) as fertilized embryos.

Techniques: shRNA

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: In vitro secondary drug screens conducted on C2C12 myoblasts and primary mouse myoblasts. (A) Proliferation assay performed with the five candidate compounds on C2C12 cells transfected with V5-tagged C774R mutant Megf10. Drug compounds were administered at 24 h of culture, and CyQUANT assays were performed at 48 h of culture. Statistically significant treatment effects are seen for sertraline (Sert) and escitalopram (Esci). The vehicle was DMSO (Veh). Horizontal bars represent the mean ± S.E.M. from 12 wells in a 96-well plate. On ANOVA, P < 0.001. On Bonferroni post hoc t-test, **P < 0.01; ***P < 0.001. (B) Proliferation assay performed with the five candidate compounds on primary myoblast cultures from wild type and Megf10−/− mice. Drug compounds were administered at 24 h of culture, and CyQUANT assays were performed at 48 h of culture. Statistically significant treatment effects are seen for sertraline and escitalopram. Scatterplots are presented, with horizontal bars representing the mean ± S.E.M. from 8 wells in a 96-well plate. On two-way ANOVA, P < 0.05. On Sidak's multiple comparisons test, *P < 0.05. (C) On migration assay, all five drug candidates yielded improved migration at 24 and 72 h, but the effect was more pronounced for sertraline and escitalopram at 72 h. Irregular yellow lines show borders between cellular and acellular zones for each image. Scale bar, 5 mm. (D) Quantifications of residual clear areas on the migration assay using ImageJ software at indicated time points. On two-way ANOVA, P < 0.05. On Sidak's multiple comparisons test *P < 0.05; **P < 0.01; n = 5 images. (E) An adhesion assay was performed on Megf10-deficient myoblasts and scrambled shRNA control myoblasts using the five candidate compounds. Megf10 shRNA-treated cells recovered significantly with Sert, Esci and Losa compounds at 60 min time points. Horizontal bars represent the mean fluorescence intensities of the adherent cells ± S.E.M. from two independent experiments, n = 2 per experiment. On two-way ANOVA, P < 0.05. On Sidak's multiple comparisons test, *P < 0.05.

Article Snippet: Megf10 mutant zebrafish (megf10 sa13029 ) contain a T to A nonsense mutation in exon 5 of zebrafish megf10 and were obtained from the Zebrafish International Resource Center (Eugene, OR) as fertilized embryos.

Techniques: In Vitro, Proliferation Assay, Transfection, Mutagenesis, CyQUANT Assay, Migration, Software, Cell Adhesion Assay, shRNA, Control, Fluorescence

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: Megf10-deficient zebrafish. Using brightfield microscopy, (A) the wild-type zebrafish maintains a straight posture, while (B) the megf10 mutant displays significant tail bending (a manifestation of severe dorsal muscle weakness). Birefringence microscopy, (C) the wild type zebrafish refracts polarized light off its dorsal muscles, whereas (D) the megf10-deficient zebrafish has black patches indicating muscle wasting, disorganized muscle fibers and myofiber degeneration pathology. Zebrafish are photographed at 4 dpf. Scale bar, 500μm. Preliminary treatment studies of sertraline and escitalopram in megf10-deficient zebrafish. Each drug cohort included 25 fish from heterozygous matings, and all fish were analyzed at 5 dpf. (E) Vehicle treated fish showed significant tail bending pathologies that were prevented with (F) 1 μm sertraline and with (G) 1 μm escitalopram. (H) Survival curves show markedly improved survival for all treatment groups. Percent of living fish were determined by detecting heartbeats. Data represent mean ± SEM relative to controls. On two-way ANOVA, P < 0.05. Sidak's multiple comparisons test results are indicated on the graph; *P < 0.05, **P < 0.01. Scale bar, 1 mm.

Article Snippet: Megf10 mutant zebrafish (megf10 sa13029 ) contain a T to A nonsense mutation in exon 5 of zebrafish megf10 and were obtained from the Zebrafish International Resource Center (Eugene, OR) as fertilized embryos.

Techniques: Microscopy, Mutagenesis, Muscles

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: Summary of three families enrolled with MEGF10 myopathy

Article Snippet: Megf10 mutant zebrafish (megf10 sa13029 ) contain a T to A nonsense mutation in exon 5 of zebrafish megf10 and were obtained from the Zebrafish International Resource Center (Eugene, OR) as fertilized embryos.

Techniques: Mutagenesis

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: Analysis of sertraline treatment in human iPSC-derived MEGF10-deficient myoblasts and murine Megf10-deficient myoblasts. (A) iPSC-derived human myoblasts representing MEGF10 deficiency (02-1, 03-1, 27-1 27-3, 27-4) showed impaired proliferation compared to asymptomatic carrier parents (02-2, 02-3) and a healthy control (H) at 48 and 72 h time points. A two-way ANOVA showed P < 0.0001. Bonferroni post test results are indicated on the graph: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (B) Human MEGF10-deficient myoblasts showed significantly improved proliferation compared to untreated myoblasts at 72 h after 1 μm sertraline hydrochloride treatment. Two-way ANOVA showed P < 0.0001. Bonferroni post-test results are depicted on the graph: *P < 0.05; **P < 0.01; ****P < 0.0001. (C) A migration assay performed on iPSC-derived human myoblasts demonstrated marked impairment in migration for MEGF10-deficient myoblasts compared to carriers and a healthy control. The yellow lines mark the borders between cellular and acellular zones. Sertraline treatment induced a marked improvement in migration patterns compared to untreated controls at 72 and 120 h. Scale bar, 5mm; +, sertraline treatment; −, no drug treatment. (D,E) Quantification of clear areas on migration assay using ImageJ software at indicated time points. On two-way ANOVA, P < 0.05. Sidak's multiple comparisons test *P < 0.05, **P < 0.01. (F) Expression levels of MEGF10, NOTCH1, HEY1, PAX7, ITGA7 and MYOD are compared via RT-PCR in MEGF10-deficient iPSC-derived myoblasts (02-1) versus carrier parent myoblasts (02-3). Data represent mean ± SEM relative to controls performed in triplicate and are presented as percentages of control myoblast expression levels. On two-way ANOVA, P < 0.05. Sidak's multiple comparisons test results are indicated on the graph; *P < 0.05, **P < 0.01; ***P < 0.001. (G) Notch1 protein expression is increased in Megf10 shRNA knockdown and wild-type C2C12 myoblasts after sertraline hydrochloride treatment, most notably at 24 and 48 h. Notch1 augmentation attenuates in the Megf10 shRNA myoblasts by 72 h. Gapdh was used as a loading control. Immunoblot images (top) and their quantification by ImageJ (bottom) are shown. The ImageJ graphs represent data from four independent experiments. Two-way ANOVA shows P < 0.001. Tukey's multiple comparison post-test results are depicted on the graph; *P < 0.05.

Article Snippet: Megf10 mutant zebrafish (megf10 sa13029 ) contain a T to A nonsense mutation in exon 5 of zebrafish megf10 and were obtained from the Zebrafish International Resource Center (Eugene, OR) as fertilized embryos.

Techniques: Derivative Assay, Control, Migration, Software, Expressing, Reverse Transcription Polymerase Chain Reaction, shRNA, Knockdown, Western Blot, Comparison

Journal: Human Molecular Genetics

Article Title: Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy

doi: 10.1093/hmg/ddz064

Figure Lengend Snippet: Sertraline treatment reactivates the Notch pathway in murine Megf10-deficient myoblasts and human MEGF10-deficient myoblasts. (A) Cell proliferation assays were performed on Megf10-deficient and wild type C2C12 myoblasts that were treated with sertraline alone, sertraline with DAPT and sertraline with serotonin. Three individual experiments with 16 wells are shown. A two-way ANOVA test was performed on different groups and conditions with Sidak’s post test, scatter plots representing the mean absorbance ± S.E.M. (****P < .0001). (B) Cell proliferation assays were performed on MEGF10-deficient human myoblasts (02-1, 03-1, 27-1) and healthy control myoblasts (H). The scatter plots indicate two independent experiments. A two-way ANOVA test was performed on different groups and conditions with Sidak’s post test, scatter plots representing the mean absorbance ± S.E.M. (C) The heat map represents mRNA expression levels measured via RT-PCR of human Notch signaling genes in human MEGF10-deficient myoblasts treated with sertraline compared to untreated healthy control myoblasts. Genes are located horizontally and samples vertically in the order given. Each sample is represented by the mean of the gene expression value of the samples within that subgroup. Healthy control is the calibrator. Blue indicates down regulation. (D) The heat map represents mRNA expression levels measured via RT-PCR of human Notch signaling genes in human MEGF10-deficient myoblasts treated with sertraline compared to untreated healthy control myoblasts. No drug treatment is the calibrator. Yellow indicates relative upregulation, and blue indicates relative downregulation. (E) Significant fold changes for the indicated genes were plotted as the mean fold change relative to the expression of the same genes in healthy untreated control subjects. Multiple unpaired t-tests were performed and P < 0.05 was considered significant.

Article Snippet: Megf10 mutant zebrafish (megf10 sa13029 ) contain a T to A nonsense mutation in exon 5 of zebrafish megf10 and were obtained from the Zebrafish International Resource Center (Eugene, OR) as fertilized embryos.

Techniques: Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression