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. 2014 Aug 1;127(15):3240–3256. doi: 10.1242/jcs.141556

Fig. 4.

Fig. 4.

The effect of Matn2 silencing and rescue on differentiation and marker gene expression of C2 myoblasts. (A) Matn2 mRNA levels were measured by QRT-PCR in proliferating C2 cell lines stably expressing Matn2 shRNA (sh7, sh4 and sh3) or the control vector (Ctrl). (B) The proliferation rate of the control and silenced cell lines was determined by using the MTS assay in growth medium. (C) Primary myotube frequency, fusion index and differentiation index of the cell lines on day 5 in differentiation medium. (D) Coding sequences are depicted for the Matn2 shRNA, the endogenous Matn2 and the rescue cDNA clone, which is resistant to shRNA interference. Immunoblotting was performed on the conditioned differentiation medium (E) and cell lysates (F) from the silenced sh3, rescued sh3-res2 and Ctrl myoblast cultures. Only the trimeric form of Matn2 is shown. Cytoskeletal β-actin served as a loading control. (G) Immunofluorescence for α-actinin-positive cells in the cultures. (H) Differentiation index as determined on day 5. (I) QRT-PCR analysis of relative gene expression (Rel. exp.) in the same cultures using TaqMan probes. Marker mRNA levels are given as fold values relative to the Hprt mRNA level. Data show the mean±s.e.m. (A–C, H). ***P<0.001, compared with control (n = 3–5). For E–G and I, representative data from three independent experiments are shown.