Journal: Nature

Article Title: Macrophage-derived glutamine boosts satellite cells and muscle regeneration

doi: 10.1038/s41586-020-2857-9

Figure Lengend Snippet: a, Schematic representation of the AAV8 expression vector for in vivo targeting of SC. U6, Pol III promoter driving the expression of the gRNA targeting the Slc1a5 locus or a non-targeting control gRNA. Since the mice used in this experiment are LSL-Cas9 x PAX7:Cre-ERT mice, Cas9 is exclusively activated in Pax7 + cells upon tamoxifen administration and, genome editing of the Slc1a5 locus will occur selectively in SC. b, Schematic overview of an AAV8-based CRISPR/Cas9-mediated in vivo genome editing. c-d, Representative images ( c ) and quantification ( d ) for Pax7 and Cas9 staining on uninjured muscles before and after tamoxifen administration ( n =4). e-f , RT-qPCR for Slc1a5 in freshly isolated SC ( n =4) ( e ) and all other mononuclear cells (non-SC) ( n =3) ( f ) upon in vivo genome editing of the Slc1a5 locus (SLC1A5-KD) specific in SC. Non-targeting control gRNA (Ctrl gRNA) was used as a control. g-h, Quantification ( g ) and representative images ( h ) of SLC1A5 and Pax7 stainings on freshly isolated SC, upon in vivo genome editing of the Slc1a5 locus (SLC1A5-KD) specific in SC ( n =3). All experiments show representative values of at least 2 independent experiments. Unpaired two-tailed t -test was everywhere applied; ns, not significant ( P >0.05). Scale bars: 50 μm ( c ); 20 μm ( h ). Graphs show mean ± SEM.

Article Snippet: Samples were then probed with mouse anti-Pax7 (DSHB, 1:20) alone or incombination with rabbit anti-SLC1A5 (Alomone Labs, 1:50) for 2h at RT followed by incubation with mouse Alexa 568 (Invitrogen, 1:10000), or a combination of mouse Alexa 488 and rabbit 568 conjugated secondary antibodies (Invitrogen, 1:1000), respectively.

Techniques: Expressing, Plasmid Preparation, In Vivo, CRISPR, Staining, Quantitative RT-PCR, Isolation, Two Tailed Test