8A). (red) and nuclei (blue) in ICAM-1+ cells treated with differentiation medium for 3 d (scale bar = 100 um). ICAM-1 was expressed by both mononuclear and multinucleated cells after treatment with differentiation medium. Because of the intense labeling of ICAM-1 in myotubes and the range of expression found in myoblasts (panel A), some of the mononuclear cells appear dimly fluorescent in the image shown. D) Representative western blot of ICAM-1 and -tubulin (loading control) in ICAM-1+ cells treated with differentiation medium for up to 6 d (5 g/lane). E) Myoblast number after 2C4 d of treatment with growth medium (n=6). F) Representative images of BrdU (red) incorporation into nuclei (blue) of control (CT), empty vector (EV), and ICAM-1+ cells at 2 d of differentiation (scale bar = 100 um). C) Quantitative analysis of the percentage of nuclei that incorporated BrdU (n=4). NIHMS632207-supplement-1.tif (3.4M) GUID:?775B02BD-5B25-4630-AC97-8A6CFC83CA24 2: Figure S2. The cytoplasmic domain of ICAM-1 in myoblast differentiation. ICAM-1+ cells were treated with vehicle, control peptide (CT-P; 100 g/ml) or ICAM-1 peptide (ICAM-1-P; 100 g/ml) at 1 d of differentiation ARHGAP1 and cell lysates were collected 2 or 24 h later. A) Representative western blot of myogenin (25 kDa) and -tubulin (loading control) after treatment with vehicle, CT-P, or ICAM-1-P. B) Quantitative analysis of western blot detection of myogenin (n=3). C) Representative western blot of phosphorylated (Thr180/Tyr182) p38 MAPK (P-p38) and B-Raf IN 1 total p38 after 2 and 24 h treatment with vehicle, CT-P, or ICAM-1-P. D) Quantitative analysis of western blot detection of phosphorylated p38 MAPK after treatment with vehicle, CT-P, or ICAM-1-P (n=3). NIHMS632207-supplement-2.tif (1.2M) GUID:?FE93756F-E39F-4518-A490-4B50583C8AB2 3: Figure S3. Expression of CD11a and CD11b. A) Representative images of CD11a and CD11b (green) and nuclei (blue) in murine leukocytes collected 5 d after intraperitoneal injection of 4% thioglycollate (positive control). Representative fluorescent images of CD11a and CD11b, as well B-Raf IN 1 as corresponding phase contrast images of control (B), empty vector (C), and ICAM-1+ (D) cells at 3 d of differentiation. NIHMS632207-supplement-3.tif (3.0M) GUID:?91F7114E-C5C0-4C41-9CA8-7BD41A3F040B 4: Figure S4. Influence of serum on myotube indices. ICAM-1+ cells were treated with differentiation medium containing 2% horse serum (serum medium) or insulin, transferrin, and selenium (serum-free medium) for up to 6 d. Quantitative analysis of myotube number (A), average number of nuclei within myotubes (B), fusion index (C), as well as myotube diameter (D), width (E), and area (F) (n=2C3). # = higher for serum-free medium compared to serum medium throughout 6 d of differentiation (main effect for medium; p<0.05). NIHMS632207-supplement-4.tif (1.8M) GUID:?62741F39-9433-4EF5-8B26-1DF33E3BD02C Abstract We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the B-Raf IN 1 ability of myoblasts to proliferate or B-Raf IN 1 differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 B-Raf IN 1 expression by skeletal muscle cells augments myogenesis, and establish.