Journal: Stem Cell Research & Therapy

Article Title: Pax7 as molecular switch regulating early and advanced stages of myogenic mouse ESC differentiation in teratomas

doi: 10.1186/s13287-020-01742-3

Figure Lengend Snippet: Analysis of basal lamina and NMJ functionality in Pax7+/+ and Pax7−/− teratomas. a – c Expression of mRNAs encoding Lama4 , Lama5 , and Lamb2 (for each genotype n = 8 to 6). d , e Expression of mRNAs encoding neuronal markers RbFox3 and Otx2 (for each genotype n = 6 to n = 7). f Immunolocalization of neurofilament (green, left column, TL transmitted light), synaptophysin (red, middle column; BTX—green; nuclei—blue), and Fasciculin (red, right column; BTX—green; nuclei—blue) in teratoma muscles. Scale bar 10 or 100 μm, as indicated. White bars—values for teratomas obtained from Pax7+/+ ESCs, gray bars—values for teratomas obtained from Pax7−/− ESCs. Data are presented as mean ± SD. Stars symbolize results of Student’s unpaired two-tailed t test: * p < 0.05; ** p < 0.01

Article Snippet: The nonspecific antibody binding sites were blocked by incubation in 3% bovine serum albumin (BSA) in PBS at room temperature for 30 min. Next, specimens were incubated in primary antibody solutions, i.e., antibodies anti: Myf5 (ab125301, Abcam; 1:100), Laminin (L9393, Sigma-Aldrich; 1:500), Laminin (L8271, Sigma-Aldrich; 1:500), skeletal muscle myosin heavy chains (skMyh, M7523, Sigma-Aldrich; 1:100), embryonic isoforms of myosin heavy chains (Myh3, F1.652, DSHB; 1:10), slow isoforms of myosin heavy chains (Myh7, BA-D5, DSHB; 1:10), fast isoforms of myosin heavy chains IIa (Myh2a, 2F7, DSHB; 1:10), fast isoforms of myosin heavy chains IIb (Myh2b, BF-F3, DSHB, 1:50), neurofilament (NF-M, 2H3, DSHB, 1:50), synaptophysin 1 (101,004, Synaptic Systems, 1:50), and AChR inhibitor fasciculin-II (F-650, Alomone Lab, Rhodamine conjugated, 7 μg/ml) diluted in 0.5% BSA in PBS, at 4 °C overnight.

Techniques: Expressing, Two Tailed Test

Journal: Frontiers in Molecular Neuroscience

Article Title: Arhgef5 Binds α-Dystrobrevin 1 and Regulates Neuromuscular Junction Integrity

doi: 10.3389/fnmol.2020.00104

Figure Lengend Snippet: Generation of muscle-specific Arhgef5 KO mice. (A) Strategy for the generation of conditional Arhgef5 knockouts. Mice with loxP sites flanking the third exon of Arhgef5 were crossed with Cre-recombinase expressing mice to produce knockout. (B) Genotyping shows successful recombination in the muscles but not tail tipss of AG5 fl/fl ; Acta-Cre mice. (C) Quantification of Arhgef5 mRNA expression in AG5 fl/fl and AG5 fl/fl ; Acta-Cre mice at 50 and 500 days of age. Results are mean ± SEM of three mice from each genotype. (D) Proper alignment of pre- and post-synaptic elements in AG5 fl/fl ; Acta-Cre muscles. Single fibers of tibialis anterior muscles isolated from AG5 fl/fl and AG5 fl/fl ; Acta-Cre mice were stained with BTX (to visualize AChR), fasciculin II (to visualize the synaptic cleft marker acetylcholinesterase), and anti-neurofilament + anti-synaptophysin antibodies (to visualize the presynaptic nerve terminal). Scale bar is 20 μm. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: For visualization of AChR we used fluorescently labeled α-bungarotoxin (B35451 or B13422; Thermo), and for visualizing the synaptic cleft, we used fasciculin II, which binds to acetylcholinesterase (F-225; Alomone Labs, labeled with FluoReporter FITC kit from Thermo).

Techniques: Expressing, Knock-Out, Isolation, Staining, Marker