Journal: bioRxiv

Article Title: Heterochronic transcription factor expression drives cone-dominant retina development in 13-lined ground squirrels

doi: 10.1101/2025.04.25.650540

Figure Lengend Snippet: Mef2c is sufficient to promote cone-specific gene expression and repress rod-specific gene expression in mouse. ( A ) Diagram of overexpression strategy used to test effects of MEF2C overexpression. ( B ) UMAP representation of P8 mouse retinal explants electroporated with a plasmid expressing GFP alone (Empty) or GFP in a bicistronic transcript with human MEF2C ( MEF2C ), (n = 7445 cell Empty, 11949 cells MEF2C ). Each point represents a single cell and is colored by cell type as determined by clustering and marker gene expression. ( C ) Heatmap of expression for select genes for cones, cone-like photoreceptor precursors, rod photoreceptor precursors, and rods in cells overexpressing MEF2C , scaled by gene. ( D ) Immunohistochemistry showing GFP and GNAT2 expression in P8 mouse retinas from Empty and MEF2C conditions. Scale bars, 50 µm. ( E ) Box plot of the number of Gnat2+, GFP+ cells divided by the total number of GFP+ cells (n = 8 for both conditions). P-values calculated by Wilcoxon rank-sum test. P0, postnatal day 0; P8, postnatal day 8; FACS, fluorescence-activated cell sorting; scRNA-Seq, single-cell RNA sequencing; ONL, outer nuclear layer; INL, inner nuclear layer; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; OE, overexpression.

Article Snippet: Primary antibodies utilized for staining 13LGS sections included goat anti-Otx2 polyclonal IgG (R&D Systems, BAF1979, 1:400) and rabbit anti-Mef2c polyclonal (Atlas Antibodies, HPA005533, 1:50).

Techniques: Gene Expression, Over Expression, Plasmid Preparation, Expressing, Marker, Immunohistochemistry, Fluorescence, FACS, RNA Sequencing

Journal: medRxiv

Article Title: Biallelic PAX7 variants cause a novel Satellite Cell-opathy with progressive muscle involvement resembling facioscapulohumeral muscular dystrophy

doi: 10.1101/2025.03.03.25322917

Figure Lengend Snippet: A. Representative images of immunolabelling for PAX7 (red) counterstained with Wheat Germ Agglutinin (WGA, white) and Hoechst (blue) showing satellite cells, fibre boundaries and nuclei respectively in different biopsies (CTRL: healthy; NAM: Necrotising Autoimmune Myopathy; FSHD STIR +ve : active; and FSHD STIR -ve : non-active disease). Percentage of PAX7 +ve nuclei (satellite cells) over the total nuclei in the section is reported per each biopsy. Green arrowheads indicate satellite cells, with representative images shown in insets. B. Schematic of retroviral infection to obtain stable cell lines, encoding for either PAX7 wild-type (PAX7wt) or PAX7 P112L-C443Y (PAX7mut), and GFP alone (empty vector; EV). RT-qPCR confirming increased PAX7 expression in both PAX7wt and PAX7 mut cell lines compared to empty vector control cells. C. Representative images of proliferating EV, PAX7wt or PAX7mut myoblast lines immunolabelled for GFP (green), PAX7 (red) and nuclei counterstained with Hoechst (white), showing clear accumulation of PAX7 in PAX7wt or PAX7mut cells. D. Quantification of PAX7 nuclear staining confirms similar accumulation and nuclear localisation in both PAX7wt or PAX7mut cell lines. E. Schematic of experimental design for differentiation analysis. Myoblasts were plated at equal numbers and induced to differentiate for 2 days, prior to immunolabelling. Representative images of myotubes from EV, PAX7wt or PAX7mut differentiated myoblasts immunolabelled for MyHC (red), GFP (green), MEF2C (magenta) and nuclei counterstained with Hoechst (white), showing severely reduced myotube formation in PAX7wt compared to PAX7mut and EV control cells. F. Example of thresholded images from E used for quantification of MyHC labelling. MyHC +ve area is dramatically reduced in PAX7wt differentiated cells, but not in PAX7mut compared to EV control. G. PAX7wt or PAX7mut myoblasts differentiate in myotube with significantly reduced diameter myotubes compared to EV control. All graphs report unpaired t-test analysis.

Article Snippet: Primary antibodies were: mouse monoclonal anti-PAX7 (Developmental Studies Hybridoma Bank; 1:500), chicken polyclonal anti-GFP (Abcam; ab13970; 1:1000), mouse monoclonal anti-MyHC (Developmental Studies Hybridoma Bank; MF20; 1:400) and rabbit polyclonal anti-MEF2C (CellSignaling; D80C1; 1:1000).

Techniques: Retroviral, Infection, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Control, Staining

Journal: Journal of translational medicine

Article Title: The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth disease-associated muscle atrophy.

doi: 10.1186/s12967-023-04016-7

Figure Lengend Snippet: Fig. 3 Ezrin was involved in myoblast differentiation/fusio. A Typical image of MyHC staining in differentiated C2C12 myoblasts treated with Ezrin overexpression or knockdown for 2, 4 and 6 days. B–D A quantitative assay for the number of MyHC + myotubes with more than 5 nuclei was performed 2, 4 and 6 days after myoblast differentiation after overexpression or knockdown of Ezrin. Three independent experiments were performed, n = 3, *P < 0.05 vs. Ad-Null; #P < 0.05 vs. Ad-Null. E RNA-seq analysis of myoblast differentiation in C2C12 myoblasts with overexpression or knockdown of Ezrin at 6 days. n = 3. F–I MyoG and MEF2c fluorescence staining and quantitative assay for myoblast differentiation. Red fluorescence indicates MyoG or MEF2c; DAPI indicates the nucleus. Three independent experiments were performed, n = 3, *P < 0.05 vs. Ad-Null; #P < 0.05 vs. Ad-Null

Article Snippet: Primary monoclonal and polyclonal antibodies against MEF2C (#5030 s, 1:200, CST), MyoG (sc-12732, 1:150, Santa Cruz), MyHC (sc-20641, 1:150, Santa Cruz), MyHC-I (NOQ, 1:200, ab234431) and MyHC-II (My32, 1:200, ab51263) were added to each well in every group and incubated for 12 h at 4 °C.

Techniques: Staining, Over Expression, Knockdown, RNA Sequencing, Fluorescence

Journal: Journal of translational medicine

Article Title: The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth disease-associated muscle atrophy.

doi: 10.1186/s12967-023-04016-7

Figure Lengend Snippet: Fig. 7 NFAT signaling is involved in the regulation of myoblast differentiation/fusion mediated by Ezrin. A RNA-seq analysis of the CaN-NFAT signaling pathway in C2C12 myoblasts with overexpression or knockdown of Ezrin at 6 days. n = 3. B Western blot of NFATc1-c4 proteins in myoblasts treated with Ezrin overexpression or knockdown for 6 days. C–F Quantitative analysis of NFATc1-c4 protein expression was performed 6 days after myoblast differentiation following Ezrin overexpression or knockdown. Three independent experiments were performed, n = 3, *P < 0.05 vs. Ad-Null; #P < 0.05 vs. Ad-Null. G Typical image of MyHC, MyoG or MEF2c staining in differentiated C2C12 myoblasts treated with Ad-shEzrin with or without Ad-NFATc1, Ad-NFATc2, Ad-shNFATc3 and Ad-shNFATc4. Red fluorescence indicates MyHC, MyoG or MEF2c; DAPI indicates the nucleus. H–J Quantitative assay for MyHC + myotubes and MEF2c + or MyoG + nuclei numbers in the above images. Three independent experiments were performed, n = 3, *P < 0.05 vs. Ad-Null; #P < 0.05 vs. Ad-shEzrin; &P < 0.05 vs. Ad-shEzrin; $P < 0.05 vs. Ad-shEzrin

Article Snippet: Primary monoclonal and polyclonal antibodies against MEF2C (#5030 s, 1:200, CST), MyoG (sc-12732, 1:150, Santa Cruz), MyHC (sc-20641, 1:150, Santa Cruz), MyHC-I (NOQ, 1:200, ab234431) and MyHC-II (My32, 1:200, ab51263) were added to each well in every group and incubated for 12 h at 4 °C.

Techniques: RNA Sequencing, Over Expression, Knockdown, Western Blot, Expressing, Staining, Fluorescence

Journal: Journal of translational medicine

Article Title: The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth disease-associated muscle atrophy.

doi: 10.1186/s12967-023-04016-7

Figure Lengend Snippet: Fig. 9 Working Model: The spatiotemporal matching pattern of Ezrin/Periaxin was involved in muscle repair in a peroneal nerve injury model. Instantaneous L-periaxin expression was highest on the 6th day, while Ezrin expression peaked on the 4th day during myoblast differentiation/ fusion (A). Ezrin promoted myoblast differentiation/fusion, myotube length and size, and myofiber specialization. L-periaxin acted as a brake for myotube length and size, negatively regulating the effect of Ezrin on myotube formation (A, B). The effects of Ezrin were related to the activated PKA-NFAT-MyoD/MEF2C signaling pathway (C). Combined gene therapy with Ezrin and L-periaxin contributed to muscle repair in a peroneal nerve injury model (D), providing a novel therapeutic strategy for the treatment of nerve injury, especially CMT4F-associated muscle atrophy

Article Snippet: Primary monoclonal and polyclonal antibodies against MEF2C (#5030 s, 1:200, CST), MyoG (sc-12732, 1:150, Santa Cruz), MyHC (sc-20641, 1:150, Santa Cruz), MyHC-I (NOQ, 1:200, ab234431) and MyHC-II (My32, 1:200, ab51263) were added to each well in every group and incubated for 12 h at 4 °C.

Techniques: Expressing