Journal: STAR Protocols

Article Title: Isolation, culture, and use of primary murine myoblasts in small-molecule screens

doi: 10.1016/j.xpro.2023.102149

Figure Lengend Snippet:

Article Snippet: bFGF Reconstitution Buffer 2 , R and D Systems , RB02.

Techniques: Plasmid Preparation, Recombinant, Modification, Cell Culture, Viability Assay, Software, Cytometry, Fluorescence, Microscopy

Journal: STAR Protocols

Article Title: Isolation, culture, and use of primary murine myoblasts in small-molecule screens

doi: 10.1016/j.xpro.2023.102149

Figure Lengend Snippet:

Article Snippet: bFGF Reconstitution Buffer 2 , R and D Systems , RB02.

Techniques: Plasmid Preparation, Recombinant, Modification, Cell Culture, Viability Assay, Software, Cytometry, Fluorescence, Microscopy

Journal: eLife

Article Title: TGF-β signaling and Creb5 cooperatively regulate Fgf18 to control pharyngeal muscle development

doi: 10.7554/eLife.80405

Figure Lengend Snippet: ( A ) UCSC binding prediction of SMAD2 binding motif to the promoter of Fgf18 gene. The boxed area indicates the binding site with the highest score. ( B ) Cut and Run analysis shows significantly more enriched Smad2/3 binding to the promoter region of the Fgf18 gene close to the predicted binding site than that of IgG in the soft palatal tissue of the control mice at E14.5. *, p-value ≤0.05. Statistical significance was assessed by unpaired t-test with two-tailed calculations. Data is presented as mean ± SEM. ( C ) qPCR analysis of Fgf18 expression in E14.5 soft palatal mesenchymal cell culture after treatment with 5 ng/ml TGF-β1 or BSA compared after 4 or 18 hr. Note the increase of Fgf18 at 4 hr is higher than at 18 hr. *, p≤0.05; ***; p≤0.001. Statistical significance was assessed by unpaired t-test with two-tailed calculations. Data is presented as mean ± SEM. ( D ) qPCR analysis of Fgf18 expression of E14.5 soft palatal mesenchymal cell culture transfected with Fgf18 or Control CRISPRi plasmid followed by treatment with 5 ng/ml TGF-β1 or BSA for 4 hr. ‘+’ or ‘-’ under the plots indicates the presence or absence of the indicated treatment. **, p≤0.01; ***, p≤0.001. Statistical significance was assessed by ANOVA. Data is presented as mean ± SEM. ( E ) Dotplot of perimysial fibroblast regulon expression pattern in individual cellular clusters of integrated scRNAseq from E13.5-E15.5 soft palatal tissue. Highlighted regulons are more enriched in the perimysial fibroblasts. ( F ) Schematic drawing of coronal section of the LVP region at E14.5. Boxed area in E indicates the region shown in G-N. The magenta and salmon colors represent the LVP myogenic cells and the other pharyngeal muscles, respectively. HB, hyoid bone; LVP, levator veli palatini; MPC, middle pharyngeal constrictor; PS, palatal shelves. ( G ) Immunofluorescence of MyoD (red) and pSMAD2 (green) in the coronal section of the LVP region at E14.5. ( H–N ) RNAScope in situ hybridization for Myod1 (red) and Fgf18 (green) ( H ), Creb5 (green) ( I ), Tbx15 (green) ( J ), Hic1 (green) ( K ), Meox1 (green) ( L ), Etv5 (green) ( M ), or Bach2 (green) ( N ) in coronal sections of the LVP region at E14.5. Arrows indicate positive signals. ( O–P ) qPCR analysis of Creb5 ( O ) and Fgf18 expression ( P ) after Creb5 siRNA treatment on E14.5 soft palatal mesenchymal cell culture compared with the control siRNA. ***, p≤0.001. Statistical significance was assessed by unpaired t-test with two-tailed calculations. Data is presented as mean ± SEM. ( Q ) qPCR analysis of Fgf18 expression following Creb5 siRNA treatment combined with 5 ng/ml TGF-β1 or BSA on E14.5 soft palatal mesenchymal cell culture, compared with the control siRNA. ‘+’ or ‘-’ under the plots indicates the presence or absence of the indicated treatment. *, p≤0.05; **, p≤0.01; ***, p≤0.001. Statistical significance was assessed by ANOVA. Data is presented as mean ± SEM. N=3 for all experiments. Tgbfr1 fl/fl or Tgbfr1 fl/+ littermates were used as controls for Osr2 Cre ;Tgfbr1 fl/fl mice. Scale bar in G indicates 100 μm in G-N. Figure 6—source data 1. Source data for . Figure 6—source data 2. Source data for . Figure 6—source data 3. Source data for . Figure 6—source data 4. Source data for . Figure 6—source data 5. Source data for . Figure 6—source data 6. Source data for .

Article Snippet: For proliferation assay, 500 ng/ml FGF18 (100–28, Peprotech) or an equal volume of BSA (RB02, R&D Systems) were added to C2C12 cells in growth medium for 1–3 days.

Techniques: Binding Assay, Control, Two Tailed Test, Expressing, Cell Culture, Transfection, Plasmid Preparation, Muscles, Immunofluorescence, RNAscope, In Situ Hybridization

Journal: eLife

Article Title: TGF-β signaling and Creb5 cooperatively regulate Fgf18 to control pharyngeal muscle development

doi: 10.7554/eLife.80405

Figure Lengend Snippet: ( A–L ) H&E staining in coronal sections of LVP region at P0.5 from control and Osr2 Cre ;Fgf18 fl/fl mice. Boxed areas in A-D and E-H are enlarged in E-H, and I-L, respectively. Black arrows point to the palatal shelf in A-D and the LVP in E-H. Green dotted line outlined the LVP in E-H. Black and white triangles point to perpendicular muscle fibers in I-L and parallel fibers in I-J. The white dotted line indicates the boundaries of perpendicular and parallel fibers in E, F, I, and J. Double-ended arrows indicate the thickness of perpendicular fibers in I and J. N=4. ( M–Q ) Immunofluorescence and quantification of MHC (red) staining on coronal section of the LVP region of control Osr2 Cre ;Fgf18 fl/fl mice at P0.5. MHC+ areas in the LVP region for quantification of cross-section areas are outlined by white dashed lines in M-P. The percentage of MHC-stained area out of the whole image area is used for quantification in Q. *, p≤0.05. N=3. Statistical significance was assessed by unpaired t-test with two-tailed calculations. Data is presented as mean ± SEM. ( R–V ) CT scanning and quantitative analysis of the muscle volume of control and Osr2 Cre ;Tgfbr1 fl/fl LVP at P0.5. A representative reconstructed control LVP from CT scanning is indicated in yellow ( R and S ) and an Osr2 Cre ;Tgfbr1 fl/fl reconstructed LVP is indicated in teal ( T and U ). **, p≤0.01. N=4. Statistical significance was assessed by unpaired t-test with two-tailed calculations. Data is presented as mean ± SEM. ( W ) A 300 μm coronal slice of the LVP region at E14 from Osr2 Cre ;Fgf18 fl/fl mouse for slice culture following bead implantation. Arrows point to BSA- or FGF18-treated bead. ( X–Y ) Immunofluorescence of MyoD (red) in the coronal section of LVP region from Osr2 Cre ;Fgf18 fl/fl mouse cultured for 3 days with BSA bead ( X ) and FGF18 bead ( Y ). White circles indicate the location of the BSA beads in X and FGF18 beads in Y. N=3. Fgf18 fl/fl or Fgf18 fl/+ littermates were used as controls for Osr2 Cre ;Fgf18 fl/fl mice. Scale bars in A, E, I, M, R, S, and W indicate 500 μm in in A-D, 100 μm in E-H, 50 μm in I-L, 400 μm in M-P, 400 μm in R and T, 100 μm in S and U, and 100 μm in W-Y, respectively. Figure 7—source data 1. Source data for . Figure 7—source data 2. Source data for .

Article Snippet: For proliferation assay, 500 ng/ml FGF18 (100–28, Peprotech) or an equal volume of BSA (RB02, R&D Systems) were added to C2C12 cells in growth medium for 1–3 days.

Techniques: Staining, Control, Immunofluorescence, Two Tailed Test, Cell Culture

Journal: eLife

Article Title: TGF-β signaling and Creb5 cooperatively regulate Fgf18 to control pharyngeal muscle development

doi: 10.7554/eLife.80405

Figure Lengend Snippet: ( A–F ) Immunofluorescence and RNAScope in situ hybridization for MyoD (red) and Caspase 3 ( A and D ), MyoD (red) and BrdU (green) ( B and E ), and Myf5 (red) and BrdU (green) (C and F) in coronal sections of LVP region at E14.5 from control and Osr2 Cre ;Fgf18 fl/fl mice. Insets are enlarged from the boxed areas. Yellow dotted line outlines the myogenic regions where the qualification analysis was performed. N=3. Fgf18 fl/fl or Fgf18 fl/+ littermates were used as controls for Osr2 Cre ;Fgf18 fl/fl mice. The schematic in the top right corner depicts the orientation and level of the section. ( G ) Quantification of the percentage of BrdU+/ My5 + double-positive cells out of the total Myf5 + cells in the LVP region from E14.5 control and Osr2 Cre ;Fgf18 fl/fl mice. **, p≤0.01. N=3. Statistical significance was assessed by unpaired t-test with two-tailed calculations. Data is presented as mean ± SEM. ( H–J ) Immunofluorescence and quantification of BrdU (red) staining on C12C12 myogenic ells following 1 day of treatment with BSA or 500 ng/ml FGF18. Proliferative rate is calculated by the percentage of BrdU+ cells out of the total number of cells. ***, p≤0.001. N=3. Statistical significance was assessed by unpaired t-test with two-tailed calculations. Data is presented as mean ± SEM. ( K ) Quantification of total number of C12C12 myogenic cells after culture for 3 days with BSA or 500 ng/ml FGF18. ***, p≤0.001. N=6. Statistical significance was assessed by unpaired t-test with two-tailed calculations. Data is presented as mean ± SEM. Scale bar in A indicates 100 μm for A-F and 30 μm for the insets in A-F; scale bar in H indicates 200 μm for H-I. Figure 7—figure supplement 2—source data 1. Source data for . Figure 7—figure supplement 2—source data 2. Source data for . Figure 7—figure supplement 2—source data 3. Source data for .

Article Snippet: For proliferation assay, 500 ng/ml FGF18 (100–28, Peprotech) or an equal volume of BSA (RB02, R&D Systems) were added to C2C12 cells in growth medium for 1–3 days.

Techniques: Immunofluorescence, RNAscope, In Situ Hybridization, Control, Two Tailed Test, Staining

Journal: BMC Microbiology

Article Title: WeFaceNano: a user-friendly pipeline for complete ONT sequence assembly and detection of antibiotic resistance in multi-plasmid bacterial isolates

doi: 10.1186/s12866-021-02225-y

Figure Lengend Snippet: Top blast hit, coverage and identity of contigs assembled by the Miniasm and Flye assemblers. For the Flye assembler, several settings, default, meta and plasmid, were tested. The samples represent 12 plasmid DNA extracts that contained multiple multidrug resistance-encoding plasmids and were published previously. *, newly assembled with the Flye assembler using the meta option; **, average coverage and identity of plasmids that could be assembled and had a BLAST hit

Article Snippet: RB02 , RB02-JN105-IncF-TET-116277-N , 116,277 , 119,228 99/97.9 , 119,956 99/97.8 , 119,986 99/97.8 , 119.936 99/96.7.

Techniques: Plasmid Preparation

Journal: BMC Microbiology

Article Title: WeFaceNano: a user-friendly pipeline for complete ONT sequence assembly and detection of antibiotic resistance in multi-plasmid bacterial isolates

doi: 10.1186/s12866-021-02225-y

Figure Lengend Snippet: Anti-microbial resistance genes and incompatibility factors identified in the assembled plasmids. *, a plasmid that could only be assembled with the Flye assembler using the meta option; anti-microbial resistance genes and incompatibility factors indicated in bold were additionally identified by WeFaceNano

Article Snippet: RB02 , RB02-JN105-IncF-TET-116277-N , 116,277 , 119,228 99/97.9 , 119,956 99/97.8 , 119,986 99/97.8 , 119.936 99/96.7.

Techniques: Plasmid Preparation