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. 2019 Dec 2;12(1):e11019. doi: 10.15252/emmm.201911019

Figure 2. IGF2R binding to CD20 is relevant for myogenic differentiation.

Figure 2

  • A–C
    Bioinformatic prediction of IGF2R and CD20 cross‐reactivity. Cartoon representation of the interaction of IGF2 (yellow) and IGF2R domain 11 complex from X‐ray structure (PDB code 2v5p). IGF2R domain 11 AB, CD, EF and HI loops and residues (shown in sticks format) involved in the hydrophobic interactions are shown (A). Cartoon representation of the IGF2R domain 11–CD20 (lime) complex obtained from docking simulations and residues involved in hydrophobic interactions is shown in stick format (B). The structure of IGF2R (in red) and IGF2 (green) is shown compared to docking poses of epitope 3. These data show that the helical region (residues 178–184) of the epitope mediates binding to the receptor as well as IGF2. The C‐terminal helix of epitope 3 partially overlaps the first α‐helix of IGF2. ClusPro‐dock IGF2R‐CD20 binding epitope poses corresponding to PDB codes 2v5p‐2oslP, 2v5p‐2oslQ and 2v5p‐3pp4P are coloured in yellow, blue and cyan, respectively (C).
  • D
    Over‐expressing CD20 in C2C12 myoblasts that co‐expressed CD20 (in green) and IGF2R (in red). DAPI‐labelled nuclei are shown in blue. Scale bars = 75 μm.
  • E
    Representative CD20 and IGF2R immunoprecipitation products immunoblotted for IGF2R and CD20, respectively, in untreated and shCD20‐treated C2C12 cell membranes and whole lysates of proteins. The immunoprecipitation output is shown as IP neg.
  • F
    Myotube immunofluorescence of cells in proliferation medium (P.M.) and after 2, 4 and 6 days of myogenic differentiation in serum‐free medium. Control (untreated), shCD20‐treated C2C12 cells, C2C12 and HSkM myoblasts pre‐treated with anti‐IGF2R for 24 h were stained. Desmin‐positive myotubes are shown in green. Scale bars = 75 μm.
  • G
    Fusion index quantification after 6 days of differentiation. One‐way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each experiment was performed in triplicate wells. All values are expressed as mean ± SEM.
  • H
    Representative WB of anti‐myogenin, anti‐Myf5, anti‐MyHC and anti‐β‐actin in total protein lysates obtained from untreated, shCD20‐treated C2C12 cells, and C2C12 and HSkM myoblasts pre‐treated with anti‐IGF2R for 24 h; cells were collected under P.M. and after 2, 4 and 6 days of myogenic differentiation. Densitometric analysis of WB data expressed as the ratio of the indicated antibody/β‐actin in arbitrary units. Two‐way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 in comparison with the results obtained in untreated cells at the corresponding time point. ## P < 0.01; ### P < 0.001; #### P < 0.0001 in comparison with the results obtained in shCD20‐treated C2C12 cells at the corresponding time point. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.

Source data are available online for this figure.