Figure 3: PJA1 marks EZH2 for proteasome-mediated degradation upon p38α activation.

(a) Western blot showing EZH2, PJA1 and tubulin levels in C2C12 cells incubated in DM after transfection with control (Scr) or siRNA against Pja1. (b) Western blot showing ubiquitin associated to immunoprecipitated EZH2 and control IgG in C2C12 cells treated as in a. MG132 was added to the cells 2 h before collecting to avoid degradation of ubiquitinated EZH2. Input represents 2% of immunoprecipitated material. (c) Co-immunoprecipitation using an antibody against EZH2 performed in C2C12 cells cultured in GM or induced to differentiate in the absence (DM) or presence (DM/SB) of SB. IgG was used as a negative control. Western blot against EZH2 and PJA1 was used to visualize interacting proteins. Input represents 10% of immunoprecipitated material. (d) Co-immunoprecipitation using an anti-MYC antibody performed in HEK293 cells transfected with the HA-tagged Pja1, HA-tagged MKK6EE and the indicated myc-tagged Ezh2 constructs. Figure shows western blot against endogenous ubiquitin and MYC and HA epitopes. Input represents 10% of immunoprecipitated material. (e) ChIP assay performed in C2C12 cells cultured in GM or induced to differentiate for 18 h in the absence (DM) or presence (SB) of SB 202190 using an antibody against H3K27me3. Mock IP is included as a negative control. Graph shows qPCR amplification of Pja1 upstream regions on immunoprecipitated material normalized against input DNA. (f) qRT-PCR showing Pja1 levels in C2C12 myoblasts cultured in the absence (GM) or presence (GM/dZNep) of dZNep as compared with cells induced to differentiate (DM). (g) Immunofluorescence using antibodies against EZH2, PJA1 and laminin on transversal sections of TA muscles derived from C57Bl6 mice, three and ten days after CTX-induced muscle injury. DAPI counter staining is also shown. Uninjured mice were used as negative control. Scale bar, 20 μm. (h) Quantification of the percentage of EZH2-positive cells per field in g. (i) Magnification of a representative image showing localization of PJA1 and EZH2 three and ten days after CTX-mediated muscle injury. Scale bar, 10 μm. Data are represented as mean±s.e.m. of three independent biological replicates. *P<0.05; **P<0.01; ***P<0.001 (Student’s test).