Journal: The FEBS journal

Article Title: GPR56 promotes myoblast fusion through SRE- and NFAT-mediated signaling but is not essential for muscle development in vivo

doi: 10.1111/febs.12529

Figure Lengend Snippet: A. Phase images of mouse myoblasts induced to differentiate over the course of 6 days (D0 – D6) illustrating the degree of myotube formation. B. Upregulation of GPR56 mRNA expression at D1 by qRT-PCR in primary mouse myoblasts which then rapidly decreases. C. Protein expression of GPR56, MyoD, myogenin, and α/β-tubulin (loading control) in myoblasts at D0 to D6, as assessed by Western blot. GPR56 protein expression peaks at D1 and rapidly decreases by D3, where little expression remains. D–E. GPR56 (green) and caveolin-1 (red) staining in differentiating primary mouse myoblasts at D1. DAPI (blue) was used to stain nuclei. Arrows point to GPR56+ cells that are positioned closely and elongated, suggesting that the cells are readying for fusion or fusing. Scale bar = 50 µm.

Article Snippet: GPR56 knockout mice (B6N.129S5- Gpr56 tm1Lex /Mmcd) were generated by Genentech/Lexicon Genetics.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Staining

Journal: The FEBS journal

Article Title: GPR56 promotes myoblast fusion through SRE- and NFAT-mediated signaling but is not essential for muscle development in vivo

doi: 10.1111/febs.12529

Figure Lengend Snippet: A. Schematic diagram showing the location of GPR56 shRNA constructs (2, 3, black arrows) against GPR56 transmembrane domains (rectangles) 2 and 4. The diamond indicates the G-proteolytic site. B. GPR56 mRNA expression by RT-qPCR in silenced C2C12s. Both shRNA2 and 3 effectively silenced the expression of GPR56. C. Western blot of GPR56, MyoD, and myogenin proteins in silenced C2C12 cells. D. Myosin Heavy Chain staining in GPR56-silenced cultures shows decreased myotube formation in GPR56 shRNA2 and 3 silenced cells. Scale bar = 50 µm. E. Fusion is decreased in GPR56-silenced cells at day 5 following differentiation. Un, uninfected; scr, scrambled oligo. * p < 0.01. F. Myotube size is decreased in GPR56-silenced cells at day 5 following differentiation. *p < 0.01. un, uninfected; scr, scrambled oligo. G. Schematic showing full-length and truncated GPR56. Diamond = G proteolytic site. Rectangles = transmembrane domains. H. Luciferase reporter assays in HEK293 cells of full-length (mGPR56, black diamond) or truncated (tGPR56, gray squares) GPR56 with luciferase reporter constructs driven by serum response element (SRE) or NFAT response element (NFAT-RE). GPR56 induces signaling from both SRE and NFAT-RE. * p<0.05. # p<0.001. n=3.

Article Snippet: GPR56 knockout mice (B6N.129S5- Gpr56 tm1Lex /Mmcd) were generated by Genentech/Lexicon Genetics.

Techniques: shRNA, Construct, Expressing, Quantitative RT-PCR, Western Blot, Staining, Luciferase

Journal: The FEBS journal

Article Title: GPR56 promotes myoblast fusion through SRE- and NFAT-mediated signaling but is not essential for muscle development in vivo

doi: 10.1111/febs.12529

Figure Lengend Snippet: A. WT and GPR56 KO mouse myoblasts undergoing differentiation at D0, D2, and D5. Scale bar = 50 µm. Green, Desmin (D0) or myosin heavy chain (D2, D5). Blue, nuclei. B. Fusion index in WT and KO differentiating mouse myoblasts. GPR56 KO myoblasts have decreased fusion at D2 and D5.* p < 0.05, n = 4. C. Overall myotube size as measured by percentage of myotubes with greater than 5 nuclei in WT and KO differentiating cultures. * p <0.05. D. Protein expression by Western blot of GPR56, MyoD, myogenin, and α/β-tubulin in differentiating myoblasts at D0-D5. GPR56 KO myoblasts show decreased MyoD expression at days 3 and 5, and increased FHL1 expression. E.GPR56 KO myoblasts (gray circles) proliferate more that WT myoblasts (black diamonds). (*p = <0.05, # = p < 0.001, n = 4 trials).

Article Snippet: GPR56 knockout mice (B6N.129S5- Gpr56 tm1Lex /Mmcd) were generated by Genentech/Lexicon Genetics.

Techniques: Expressing, Western Blot

Journal: The FEBS journal

Article Title: GPR56 promotes myoblast fusion through SRE- and NFAT-mediated signaling but is not essential for muscle development in vivo

doi: 10.1111/febs.12529

Figure Lengend Snippet: A. Representative coronal flair MRI image from an unaffected individual (NORMAL) and coronal T2 images from individuals with confirmed mutations in GPR56 and POMGnT1. Patients exhibit enlarged ventricles (asterisks), presence of diffused cortical abnormalities (white arrowhead) and presence of cerebellar abnormalities, including a small vermis in the GPR56 patient (arrow). B. Serum creatine kinase levels and motor developmental delays in patients with BFPP. ND: Not determined. C. H&E staining of one-month-old gastrocnemius (top, GA) and tibialis anterior (bottom, TA) muscles shows no difference between wildtype and knockout muscle. Scale bars = 50 µm. D. Myofiber diameter in TA muscle shows no difference between WT and KO. E. Serum CK levels in WT and KO mice shows slightly elevated serum CK levels in knockout mice. *p=0.012, n = 11–12. F. mRNA expression in WT and KO gastrocnemius muscle. Expression of MyoD, FHL1, NFATc2, and NFATc3 are decreased in KO muscle. * p<0.05, n = 6.

Article Snippet: GPR56 knockout mice (B6N.129S5- Gpr56 tm1Lex /Mmcd) were generated by Genentech/Lexicon Genetics.

Techniques: Staining, Muscles, Knock-Out, Expressing

Journal: The FEBS journal

Article Title: GPR56 promotes myoblast fusion through SRE- and NFAT-mediated signaling but is not essential for muscle development in vivo

doi: 10.1111/febs.12529

Figure Lengend Snippet: A. H&E staining of GPR56 WT and KO gastrocnemius muscle at days 4, 6, and 18 after cardiotoxin injury. KO morphology and timing does not look different from WT. B. mRNA expression of GPR56 by RT-qPCR shows transient upregulation of GPR56 during regeneration. C. Myofiber diameter in cardiotoxin-injured WT and GPR56 KO gastrocnemius muscle shows no difference in diameter between WT and KO. D. mRNA expression by RT-qPCR of various genes in WT (black diamond) and GPR56 KO (gray circle) cardiotoxin-injured muscle. Myf5, MyoD, and myogenin are delayed in expression in KO muscle. * p<0.05, n = 3. E. Sample Western blots of myosin heavy chain protein expression. F. Quantification of the amount of MHC I, IIA, or IIB protein expression by Western blot in WT and KO gastrocnemius muscle in mice of various ages shows no difference in the amount of MHC isoforms between WT and KO. G. Quantification of the % of positive MHC I, IIA, or IIB fiber types in KO versus WT muscles, based on immunofluorescence staining in 4 littermate pairs.

Article Snippet: GPR56 knockout mice (B6N.129S5- Gpr56 tm1Lex /Mmcd) were generated by Genentech/Lexicon Genetics.

Techniques: Staining, Expressing, Quantitative RT-PCR, Western Blot, Muscles, Immunofluorescence