Journal: Cell reports

Article Title: The adhesion G-protein-coupled receptor Gpr116 is essential to maintain the skeletal muscle stem cell pool.

doi: 10.1016/j.celrep.2022.111645

Figure Lengend Snippet: Figure 5. GPR116 interacts weakly with Gqa and with b-arrestin upon Stachel peptide stimulation (A) Scheme of BRET-based assays to reveal GPCR G protein coupling mechanisms. GPR116, Gb, Gg-GFP, and Ga-Rluc are indicated. (B–E) SDP116 concentration response curves for activation of (B) Gqa-Rluc, (C) Gsa-Rluc, (D) G12a-Rluc, or (E) Gia-Rluc BRET biosensors. (F) Scheme of enhanced bystander BRET-based assays to reveal GPCR b-arrestin coupling at the plasma membrane. GPR116, CAAX-GFP, and b-arrestin-Rluc are indicated. (G–H) SDP116 concentration response curves for activation of (G) Arrb1-Rluc (b-arrestin1-Rluc) or (H) Arrb2-Rluc (b-arrestin2-Rluc). (I and J) SDP116 and scrambled peptide concentration response curves for activation of (I) Arrb1-Rluc (b-arrestin1-Rluc) or (J) Arrb2-Rluc (b-arrestin2-Rluc).

Article Snippet: Primary antibodies were against PAX7 (DSHB), MYOD (SantaCruz, sc-304 and sc-377460), DYSTROPHIN (Pierce, PA137587), GPR116 (Invitrogen PA5-75810), b-arrestin1 (CST 12697S) Laminin (Sigma L9393), MYOG (Abcam 124800), DDX6 (Bethyl A300461A), embryonic Myosin Heavy Chain (F1.652, DSHB), Myosin Heavy Chain (MF20, DSHB), CREB (Cell Signaling 86B10) and b-ACTIN (Sigma A5441).

Techniques: Concentration Assay, Activation Assay, Clinical Proteomics, Membrane

Journal: Cell reports

Article Title: The adhesion G-protein-coupled receptor Gpr116 is essential to maintain the skeletal muscle stem cell pool.

doi: 10.1016/j.celrep.2022.111645

Figure Lengend Snippet: Figure 6. b-arrestin1 is expressed in quiescent MuSCs, where it is localized to the nucleus and required for the activity of SDP116 to prevent MuSC activation (A and B) Relative (A) Arrb1 and (B) Arrb2 levels, determined by qRT-PCR, in MACS isolated MuSCs isolated from uninjured TA muscle (no injury), and 1, 4 and 21 days post ctx injury (dpi). Levels are reported normalized to Actb. ***p < 0.001, **p < 0.01, ns, not significant. (C) Relative Arrb1 and Arrb2 levels, determined by qRT-PCR, in fresh MACS isolated MuSCs from wild-type and Dmdmdx mice. Levels are reported normalized to Actb. (D) Immunofluorescence with antibodies against PAX7 (green) and b-arrestin1 (red) on transverse sections of TA muscle. Scale bars represent 50 mm. Arrows (white) indicate position of PAX7(+) MuSCs with nuclear localized b-arrestin1.

Article Snippet: Primary antibodies were against PAX7 (DSHB), MYOD (SantaCruz, sc-304 and sc-377460), DYSTROPHIN (Pierce, PA137587), GPR116 (Invitrogen PA5-75810), b-arrestin1 (CST 12697S) Laminin (Sigma L9393), MYOG (Abcam 124800), DDX6 (Bethyl A300461A), embryonic Myosin Heavy Chain (F1.652, DSHB), Myosin Heavy Chain (MF20, DSHB), CREB (Cell Signaling 86B10) and b-ACTIN (Sigma A5441).

Techniques: Activity Assay, Activation Assay, Quantitative RT-PCR, Isolation

Journal: Cell reports

Article Title: The adhesion G-protein-coupled receptor Gpr116 is essential to maintain the skeletal muscle stem cell pool.

doi: 10.1016/j.celrep.2022.111645

Figure Lengend Snippet: Figure 7. GPR116 signaling promotes b-arrestin1:CREB nuclear interactions in quiescent MuSCs (A) Single EDL myofibers isolated from wild-type and Arrb1/ mice, immunolabeled for MuSCs with antibodies against PAX7 (green) along with b-arrest- in1:CREB PLA (red). Nuclei are counterstained with DAPI (merged). (B) 24- to 48-h-cultured single EDL myofibers immunolabeled for activated MuSCs with antibodies against MYOD (green) along with b-arrestin1:CREB PLA (red). Nuclei are counterstained with DAPI (merged). Scale bars in (A) and (B) represent 5 mm. (C) Average numbers of b-arrestin1:CREB PLA puncti in nuclei of MuSCs identified on wild-type single EDL myofibers in (A) and (B). Mean and SEM from three independent mice (n R 200 myofibers). *p < 0.05, ns, not significant. (D) Single EDL myofibers isolated from tmx-administered Pax7CreERT2/+ and Pax7CreERT2/+; Gpr116fl/flmice, immunolabeled for MuSCs with antibodies against PAX7 (green) along with b-arrestin1:CREB PLA (red). Nuclei are counterstained with DAPI (merged). Scale bars, 5 mm. (E) Average number of b-arrestin1:CREB PLA puncti in nuclei of MuSCs identified in (D). Mean and SEM from three independent mice (n R 200 myofibers). *p < 0.05.

Article Snippet: Primary antibodies were against PAX7 (DSHB), MYOD (SantaCruz, sc-304 and sc-377460), DYSTROPHIN (Pierce, PA137587), GPR116 (Invitrogen PA5-75810), b-arrestin1 (CST 12697S) Laminin (Sigma L9393), MYOG (Abcam 124800), DDX6 (Bethyl A300461A), embryonic Myosin Heavy Chain (F1.652, DSHB), Myosin Heavy Chain (MF20, DSHB), CREB (Cell Signaling 86B10) and b-ACTIN (Sigma A5441).

Techniques: Isolation, Immunolabeling, Cell Culture