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: Journal of Biological Chemistry

Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

doi: 10.1074/jbc.ra119.008544

Figure Lengend Snippet: Figure 1. (A) Immunoblot analysis of endogenous G0S2 protein half-life in neonatal rat cardiomyocytes. Cells were treated with cycloheximide (CHX) (100 µg/ml) at the indicated times. (B) Immunoblotting of cardiomyocytes treated with proteasome inhibitors (MG132: 10 µM; Lactacystin: 10 µM; PS-341: 0.3 µM) or lysosomal inhibitors (chloroquine: 100 µM; NH4Cl: 10 mM). Four hours after the treatment, cells were harvested and subjected to immunoblot analysis. (C) Schematic workflow of siRNA library screening. C2C12 cells stably expressing EGFP-fused G0S2 (C2C12/EGFP-G0S2 cells) or EGFP-fused CL1 degron peptide (C2C12/EGFP-CL1 cells) were transfected with the indicated siRNA. Seventy-two hours after transfection, cells were imaged and fluorescence intensities of EGFP were analyzed by IN Cell Analyzer 6000. See also Fig. S1. (D) Distribution of siRNAs ranked according to values of log2 fold change (FC) of EGFP-G0S2 intensity that is averaged and normalized to the siCTL in three independent experiments. Black and red dots represent negative and positive hits, respectively. (E) Scatter plot of the log2FC of EGFP intensity in C2C12/EGFP-CL1 cells and the number of C2C12/EGFP-G0S2 cells, both averaged and normalized to the siCTL in three independent experiments. Each dot represents one siRNA pool targeting one gene. (F) C2C12 cell lines stably expressing HA-tagged G0S2 (C2C12/HA-G0S2 cells) were transfected with 30 nM of either siCTL or siRNF126, as indicated. After 48 h incubation, total RNA was extracted and analyzed by qPCR. Data represent mean values ± S.D. (n=3). ***P<0.001. (G) Seventy-two hours after transfection of siRNA as in (F), cells were harvested and subjected to immunoblot analysis. RNF126 was detected by a RNF126 #A antibody.

Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

Techniques: Western Blot, Library Screening, Stable Transfection, Expressing, Transfection, Fluorescence, Incubation

Journal: Journal of Biological Chemistry

Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

doi: 10.1074/jbc.ra119.008544

Figure Lengend Snippet: Figure 4. (A–E) The indicated siRNA was expressed for 72 h in (A) cardiomyocytes or (B–E) C2C12/HA-G0S2 cells. Cells were harvested and subjected to immunoblotting. (C) Cells were solubilized and ubiquitinated proteins were enriched by TUBE pull-down assay. (D) Immunoblotting of cells that were treated with cycloheximide at the indicated times (0–120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0min) and is shown graphically. The asterisks denote statistical significance comparing siCTL- and siBAG6-treated cells. Data represent mean values ± S.D. (n=3). *P<0.05. **P<0.01. (F) Representative YFP/CFP ratiometric pseudocolored images of Mit-ATeam fluorescence in cardiomyocytes expressing the indicated siRNA and adenoviral Mit-ATeam for 48 h. (Scale bar, 10 µm) (G) YFP/CFP emission ratio plots of Mit-ATeam fluorescence in cardiomyocytes transfected siCTL (n=18), siBAG6 #1 (n=20) or siBAG6 #3 (n=20) during hypoxia. All of measurements were normalized to the ratio at time 0 and compared between cardiomyocytes with siCTL, siBAG6 #1 and #3 at each time point. Data represent mean values ± SEM. n.s., not significant.

Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

Techniques: Western Blot, Pull Down Assay, Fluorescence, Expressing, Transfection

Journal: Journal of Biological Chemistry

Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

doi: 10.1074/jbc.ra119.008544

Figure Lengend Snippet: Figure 5. (A) Schematic diagram of generated mutants. (B) C2C12 cells were transfected with the indicated G0S2 mutants. After 4 h treatment with DMSO (D : 0.1%) or MG132 (MG : 10 µM), cells were harvested and subjected to immunoblot analysis. (C) C2C12/HA-G0S2 WT or E44A cells were solubilized and ubiquitinated proteins were enriched by TUBE2 pull-down assay. (D) C2C12/HA-G0S2 WT or E44A cells were treated with cycloheximide at the indicated times (0-120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0 min) and is shown graphically. The asterisks denote statistical significance comparing C2C12/HA-G0S2 WT and E44A cells. Data represent mean values ± S.D. (n=3). **P<0.01. (F) Immunoprecipitation of HA-G0S2 in HEK293T cells. Cells expressing HA-tagged G0S2 WT or E44A were harvested and immunoprecipitated with anti-HA antibody. (G) C2C12 cells expressing HA-G0S2 WT or E44A were transfected with the indicated siRNA for 48 h. After solubilizing with the buffer containing 1% Triton X-100, lysates were centrifuged and the supernatant (S) and pellet (P) fractions were analyzed by immunoblotting. The pellet contains the detergent-insoluble protein aggregates. GAPDH and vimentin are used as the detergent-soluble and -insoluble fractions, respectively.

Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

Techniques: Generated, Transfection, Western Blot, Pull Down Assay, Immunoprecipitation, Expressing

Journal: Advanced functional materials

Article Title: Light-controlled growth factors release on tetrapodal ZnO-incorporated 3D-printed hydrogels for developing smart wound scaffold

doi: 10.1002/adfm.202007555

Figure Lengend Snippet: (A) Live/Dead and F-Actin/DAPI staining on C2C12 behaviors cultured on the t-ZnO-laden GelMA hydrogels with and without VEGF coating at various concentrations of t-ZnO. (Β) Quantified viability of cultured C2C12 cells on the constructs with and without VEGF coating and different concentrations of t-ZnO. (n = 3, 2 random images/sample, *p < 0.05, ***p < 0.001). (C) Quantified cell covered area that was calculated from F-Actin/DAPI staining images of cultured C2C12 cells on the t-ZnO-laden GelMA hydrogels obtained on day 3 of culture. (n = 3, 2 random images/sample, *p < 0.05, ***p < 0.001). (D) Quantified viability of cultured HUVECs on the t-ZnO-laden GelMA hydrogels with and without VEGF coating at various concentrations of t-ZnO on day 1 of culture. (E) PrestoBlue™ assay of HUVECs seeded on different concentrations of t-ZnO and t-ZnO VEGF-coated hydrogels over 7 days of culture. (n = 3). (F) Higher CD31 covered area observed on VEGF-coated t-ZnO-laden GelMA hydrogels which were calculated from CD31/DAPI staining images obtained on day 7 of culture. (n = 5, *p < 0.05, ***p < 0.001). (G) Representative fluorescence microscope images of live/dead, F-Actin/DAPI, and CD31/DAPI staining for cultured HUVECs on t-ZnO-laden GelMA hydrogels with and without VEGF coating at various concentrations of t-ZnO on day 7 of culture.

Article Snippet: To culture the needed cells, vials of C2C12 and HUVECs were purchased from Lonza Bioscience, along with the corresponding Dulbecco’s modified eagle medium (DMEM) and Endothelial Cell Basal Medium-2 (EBM-2).

Techniques: Staining, Cell Culture, Construct, Prestoblue Assay, Fluorescence, Microscopy

Journal: Cancer Metastasis Reviews

Article Title: The developing story of Sprouty and cancer

doi: 10.1007/s10555-014-9497-1

Figure Lengend Snippet: Responses of different cell types to the Sprouty-induced regulation reported by different investigators

Article Snippet: Tsumura et al. [ ] , 4 , , C2C12 , , × , , , , , , , Cofilin.

Techniques: