Expression of mRNAs encoding integrin alpha3 and other adhesion proteins at 24 and 48 hours after transfection of MPCs-derived myoblasts with siRNA-alpha3. sqRT-PCR analysis.

<p>NT – control, not transfected myoblasts, siRNA-alpha3 – myoblasts transfected with siRNA downregulating the expression of integrin alpha3. Optical densities of representative bands are shown as a percentage of GAPDH band density taken as a 100%.</p

Downregulation of alpha3 integrin expression reduces fusion of MPCs-derived myoblasts.

<p>A - Pappenheim's staining of fusing myoblasts. B – total number of nuclei calculated at day 12 of myoblast culture. C –index of fusion analyzed at day 12 of culture in control and experimental myoblasts shown as a percentage of myotube nuclei per number of all nuclei. D - proportion of 2–4, 5–7, 8–10 and >11 nuclear myotubes per number of all myotubes, respectively. NT – control, not transfected myoblasts, TR – control myoblasts cultured in medium supplemented with transfection reagent, siRNA-cont - control myoblasts transfected with scramble siRNA, siRNA-alpha3 – myoblasts transfected with siRNA downregulating the expression of integrin alpha3. Error bars indicate SEM, results were analyzed by Kruskal-Wallis test. Kruskal-Wallis One Way Analysis of Variance showed differences between experimental groups which were considered statistically significant when p<0.05 (marked with asterisks). * p≤0.05.</p

Downregulation of integrin alpha3 reduces the C2C12 ability to fuse with control myoblasts.

<p>A – control - myoblasts transfected with scramble siRNA-AlexaRed (red) cultured with myoblasts stained with QTracker (green), experiment+siRNA-alpha3– myoblasts cotransfected with scramble siRNA-AlexaRed (red) and siRNA-alpha3 cultured with control myoblasts stained with QTracker (green). Hybrid myotubes are yellow. Scale bars 20 µm. B –number of hybrid myotubes formed by control myoblasts transfected with scramble siRNA-AlexaRed and control myoblasts stained with QTracker (control) compared with the number of hybrid myotubes formed by myoblasts co-transfected with scramble siRNA-AlexaRed and siRNA-alpha3 and control myoblasts stained with QTracker (siRNA-alpha3) (day 11 of culture). Error bars indicate SEM, results were analyzed by Student's test and differences were considered statistically significant when p<0.05 (marked with asterisks). ** p≤0.01.</p

Changes in expression and localization of adhesion proteins during <i>Soleus</i> muscle regeneration.

<p>A - level of mRNAs encoding adhesion proteins analyzed by sqRT-PCR. Optical densities of representative bands are shown as a percentage of GAPDH band density taken as a 100%. B – localization of integrin alpha3 (red) in intact and in regenerating muscle at day 3, 5, 7, 14. Nuclei – blue. White arrows show degenerating myofibers. Scale bars 50 µm.C –colocalization of Pax7, MyoD, myogenin (MG) (red) with integrin alpha3 (green) in intact muscle and at day 1, 3 and 5 of regeneration (intact, d1, d3, d5 respectively). White arrows - myoblasts, pink – muscle fiber nuclei, yellow - MyoD, myogenin and integrin alpha3 negative cells. D – control staing with secondary antibodies only. Nuclei – blue. Scale bars 50 µm. E - localization of integrin beta1, ADAM12, CD9, or M-cadherin (green) at day 3 of regeneration. Nuclei – blue. Scale bars 50 µm. F – immunoblotting analysis of M-cadherin (M-cad), integrin beta1, ADAM12 (AD12), CD9 and integrin alpha3 level during muscle regeneration (days 1, 3, 7, 14).</p