Journal: bioRxiv

Article Title: Vitronectin defines a differentiation-restraining extracellular matrix state in myogenic cells

doi: 10.64898/2026.01.14.699461

Figure Lengend Snippet: a, Schematic representation of the myogenic differentiation processes in C2C12 myoblasts dependent on culture medium and expression dynamics of representative muscle differentiation markers. Proliferation of myoblasts requires growth medium (GM; 10% fetal bovine serum (FBS) supplemented medium), whereas differentiation medium (DM; 2% horse serum (HS) supplemented medium) induces rapid differentiation into multinucleated myotubes and loss of proliferative capacity. b, Representative phase contrast and immunofluorescence images showing expression of the differentiation marker Myogenin on day 3 in DM. White arrowheads indicate representative differentiating myotubes. Differentiation index quantified by the proportion of myogenin-positive cells relative to total nuclei is shown in the graph. (Control: n = 1652, Vitronectin: n = 1746) c, Time-course observation of myotube differentiation by immunofluorescence for myosin heavy chain (MHC). d, Fusion index quantified by the number of nuclei per MHC-positive myotube on day 3 in DM shown in the graph (Control: n=50, Vitronectin: n=50 in MHC-positive cells). The numbers shown in the immunofluorescence images indicate of nuclei in the representative MHC-positive myotube. e, Immunoblot analysis of C2C12 cells for the representative myogenic differentiation markers: MyoD, Myogenin, and MHC. f, Immunoblot analysis of C2C12 cells for non- and phosphorylated form of STAT3, ERK1/2, and p38 MAPK. g-h , Immunofluorescence analysis of MHC expression after 3 days in DM under the indicated conditions: control, VN, VN plus cRGDfV, and cRGDfV alone. Quantification shows the proportion of MHC-positive cells relative to total nucle as differentiation indexi (g) and fusion index (h). Scale bar: 100 µm. Nuclei were counterstained with DAPI. Data in the graphs are presented as mean ± s.d. Statistical significance determined using Student’s t-test; ** p < 0.01 .

Article Snippet: As a first antibody, Anti-MyoD (5.8A, 1:5000; Novus Biologicals, NB100-56511), anti-Myogenin (1:10000; Abcam, ab124800), Anti-MHC (MF20, 1:5000; R&D Systems, MAB4470), Anti-β-Actin (C4, 1:5000; Santa Cruz, sc-47778), Anti-Stat3 (D3Z2G, 1:5000; Cell Signaling Technology, #12640), Anti-phospho-Stat3 (Tyr705, D3A7, 1:5000; Cell Signaling Technology, #9145), Anti-ERK1/2 (C-9, 1:5000; Santa Cruz, sc-514302), Anti-phospho-ERK (E-4, 1:5000; Santa Cruz, sc-7383), Anti-p38α/β MAPK (A-12, 1:5000; Santa Cruz, sc-7972), anti-phospho-p38 (E-1, 1:5000; Santa Cruz, sc-166182), Anti-p21 Waf/Cip1 (F-5, 1:5000; Santa Cruz, sc-6246), Anti-p27 Kip2 (F-8, 1:5000; Santa Cruz, sc-1641), Anti-FGF Receptor 1 (D8E4, 1:5000; Cell Signaling Technology, #9740), Anti-p-FGF Receptor 1 (Try653/654) (D4X3D, 1:5000; Cell Signaling Technology, #52928), Anti-Insulin Receptor β (4B8, 1:5000; Cell Signaling Technology, #3025), Anti-p-IGF-1 Receptor β (Tyr1135/1136)/Insulin Receptor β (Try1150/1151) (19H7, 1:5000; Cell Signaling Technology, #3024), Anti-Vitronectin 65/75 (D-8, 1:5000; Santa Cruz, sc-74484), Anti-p16 INK4A (F-12, 1:5000; Santa Cruz, sc-1661), p-χHistone H2A.X (Ser 139, 1:5000; Santa Cruz, sc-517348).

Techniques: Cell Characterization, Expressing, Immunofluorescence, Marker, Control, Western Blot