The LIM domain protein nTRIP6 modulates the dynamics of myogenic differentiation

Figures

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  Figure 1. nTRIP6 expression is transiently increased during myoblast differentiation. C2C12 myoblasts lysates harvested at the indicated day of a differentiation experiment—day 0 corresponds to the switch from growth medium (GM) to differentiation medium (DM)—were subjected to Western blotting using the indicated antibodies. (a) Representative blots are shown. Full-length blots are presented in Supplementary Fig. S1. (b) The expression of TRIP6 and nTRIP6, as well as of MYOG and TNNI2 relative to the β-actin loading control are presented as mean ± SD of three independent experiments. Individual values are depicted by symbols, each representing an independent experiment.

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  Figure 2. Blocking nTRIP6 function accelerates early and delays late differentiation. Control untransfected C2C12 cells (Co) or cells transfected with either the mCherry-tagged, nuclear targeted nTRIP6 blocking peptide (BP) or the control, scrambled version of the peptide (cBP) were subjected to a differentiation experiment. (a) Twenty-four hours after transfection cell nuclei were counterstained with DRAQ7 and imaged by confocal microscopy. Representative images are shown. (b,c) Cells were harvested at the indicated day and the relative levels of the Myog (b) and Tnni2 (c) mRNAs were determined by reverse transcription and real-time PCR. Results are plotted relative to the expression of the Rplp0 gene (mean ± SD of three independent experiments). Bonferroni corrected P values are for (b) α = 0.044, β = 0.027 and γ = 0.012 and for (c) α = 0.050, β = 0.053 and γ = 0.047. (d–f) C2C12 cells transfected with the BP or the cBP were harvested at the indicated day of a differentiation experiment. Cell lysates were subjected to Western blotting using the indicated antibodies. Representative blots are shown. Full-length blots are presented in Supplementary Fig. S5. The expression of MYOG, TNNI2 and…

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  Figure 3. Blocking nTRIP6 function does not affect myoblast proliferation and cell cycle exit. C2C12 cells transfected with either the control (cBP) or the blocking peptide (BP) were subjected to a differentiation experiment. (a) Cells were counted at the indicated day and results are shown relative to the cBP-transfected cells at day-2 and are mean ± SD of three independent experiments. Individual values are depicted by symbols, each representing an independent experiment. (b, c) Cells were pulsed with EdU for 1 h at the indicated time point, fixed, stained for EdU incorporation and peptide expression (mCherry) and analysed by confocal microscopy. The percentage of EdU-positive nuclei among transfected cells (mCherry positive) is presented as mean ± SD of three independent experiments (b). Individual values are depicted by symbols, each representing an independent experiment. Representative images are shown (c, scale bar: 200 µm).

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  Figure 4. Blocking nTRIP6 function delays late differentiation and fusion. Control C2C12 cells transfected with either the mCherry-tagged, nuclear targeted nTRIP6 blocking peptide (BP) or the control, scrambled version of the peptide (cBP) were subjected to a differentiation experiment. Cells were fixed at the indicated day and subjected to immunofluorescence analysis using antibodies against MYH3 and mCherry. Nuclei were counterstained using DAPI. (a) Representative images are presented (scale bar 100 µm). (b) The percentage of MYH3 expressing cells among transfected cells (mCherry-positive), (c) the fusion index (percentage of mCherry positive nuclei within fused myotubes), and (d) the number of mCherry-positive nuclei per myofiber are presented as mean ± SD of three independent experiments.

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  Figure 5. Recombination efficiency in satellite cells and loss of TRIP6/nTRIP6 expression in Trip6scko myogenic cells. Myofibres with the associated satellite cells isolated from the Extensor digitorum longus of Trip6fl/fl and Trip6scko mice were subjected to immunofluorescence analysis using anti-TRIP6/nTRIP6 and anti-PAX7 antibodies. Nuclei were counterstained using DRAQ5. (a) Representative images are shown (scale bar: 20 µm). (b) The percentage of satellite cell (PAX7-positive) showing TRIP6 / nTRIP6 immunoreactivity is presented as mean ± SD (3 animals per genotype, 10 myofibres per animal).

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  Figure 6. Skeletal muscle regeneration in Trip6 knockout mice. (a) Schematic representation of the protocol. The Trip6 gene was knocked out in satellite cells by intraperitoneal injection of tamoxifen in Trip6fl/fl; Pax7Cre-ERT2/wt mice and muscle degeneration was induced by injection of notexin in the M. soleus. (b-g) The regenerating and uninjured contralateral muscles of Trip6scko (ko) and Trip6fl/fl control animals (fl/fl) were harvested at the indicated day post-injury (dpi). Muscle sections were stained with anti-Laminin, anti-MYOD and anti-Ki67 antibodies and counterstained with DAPI. (b) Representative images at 14 dpi are shown (the full set is presented in Supplementary Fig. S6; scale bar: 30 µm). (c) Number of MYOD-positive mononuclear cells normalized to the number of myofibres; (d) Number of MYOD-positive mononuclear cells expressing Ki67 normalized to the number of myofibres; (e) Number of MYOD-positive mononuclear cells not expressing Ki67 normalized to the number of myofibres; (f) distribution of the number of centrally located nuclei detected on myofibres cross-sections; (g) Minimum Feret’s diameter of the regenerating myofibres expressed as percent of the contralateral…

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  Figure 7. Expression of myosin heavy chains. Sections of regenerating Trip6scko (ko) and Trip6fl/fl control (fl/fl) muscles harvested at the indicated day post- injury (dpi) were stained with an anti-Laminin antibody (cyan) together with either an anti-MyHC1 or an anti-MyHC2a antibodiy (magenta) as indicated. (a) Representative images are shown (scale bar: 250 µm). (b) The number of myofibres positive for MyHC1 and MyHC2a is presented as percent of the total number of myofibres. Bonferroni corrected P values are presented (5 animals per group).