Figure 3: Nuclear Linc-RAM physically interacts with MyoD in the muscle cells.

(a,b) Linc-RAM in cytoplasmic (Cyto), nuclear-soluble (Nuc.Sol) and nuclear-insoluble (Nuc.Insol) fractions of proliferating (a) and differentiating (b) C2C12 cells was determined by qRT–PCR. Neat1 (nuclear paraspeckle assembly transcript 1) were used as markers for the nuclear fraction; GAPDH was used as markers for the cytoplasmic fraction. The data are representatives of three independent experiments. (c) Subcellular localization of Linc-RAM in differentiated and undifferentiated C2C12 cells (DM 2 days) was examined by RNA FISH using a pool of singly-Cal610-labeld ODN probes against the Linc-RAM (Linc-RAM Probes). A pool of singly-Cal610-labeld ODN probes against the EGFP-coding sequence served as nonsense control (control probes). The nuclei were stained with DAPI. The images are representatives of three independent experiments. Scale bar, 20 μm. (d) RNA immunoprecipitation (RIP) was used to examine the physical interaction of Linc-RAM with MyoD. Muscle homogenates were immunoprecipitated using anti-MyoD antibodies, and Linc-RAM in immunoprecipitates were detected by semi-qRT–PCR (left) and qRT–PCR (right). GAPDH served as a negative control. MyoD in above immunoprecipitates were detected by western blotting. (e) The direct interaction between MyoD and Linc-RAM was examined by electrophoresis mobility shift assay (EMSA) with purified GST–MyoD fusion protein. The presented blot is a representative of three independent experiments. (f) The interaction between MyoD and different truncated form of Linc-RAM were examined by EMSA. The presented blot is a representative of three independent experiments.