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. Author manuscript; available in PMC: 2010 Oct 9.
Published in final edited form as: Mol Cell. 2009 Oct 9;36(1):61–74. doi: 10.1016/j.molcel.2009.08.008

Figure 4. miR-214 Reduces Ezh2 Protein and Accelerates Muscle Cell Differentiation.

Figure 4

(A) miR-214 transcripts (left panel) and time course of protein expression of Ezh2, MHC, myogenin and tubulin (right panel) in empty vector or miR214-wt retrovirally-transduced C2C12 cells. Quantification of the band corresponding to Ezh2 protein was performed with the ImageJ software program. (B) Ezh2 mRNA in empty vector or miR-214-wt C2C12 cells. (C) MHC immunostaining of C2C12 cells transduced with empty vector, miR-214-wt, or mir-214-mut and differentiated for 24 hrs. The nuclei were visualized with DAPI. The fusion index was determined by counting of the number of nuclei present in MHC+ cells per number of total nuclei and expressed as percentage. Error bars represent the standard deviations. (D) Immunoblot of Ezh2, myogenin, and tubulin in C2C12 cells transduced with empty vector, miR-214-wt, or mir-214-mut and differentiated for 12 hrs. (E) Immunoblot of Ezh2, MHC, myogenin and tubulin in C2C12 cells transfected with Ezh2 siRNAs (left panel) and immunoblot of Ezh2, GAPDH, and myogenin mRNA in Ezh2 flox/flox primary myoblasts infected with adenoviruses expressing Cre recombinase (right panel). (F) Immunoblot of Ezh2, MHC, myogenin and tubulin in C2C12 myoblasts transfected without (-), or with scrambled or anti-miR-214 oligomers. Bands corresponding to Ezh2 protein were quantified using the NIH Image J software. (G) Immunoblot of Ezh2, Myc, myogenin and tubulin of extracts derived from C2C12-expressing wt or mutated miR-214 cells transfected with pCDNA3 (empty vector) or pCDNA3-Myc-tagged-Ezh2 vector, and differentiated for 12 hrs. The signal corresponding to the endogenous Ezh2 protein in C2C12 cells expressing exogenous Ezh2-myc and miR-214 WT was reduced by ∼ 40% when compared to control. The miR-214 mutant had no effect on endogenous Ezh2. Quantification was performed with NIH ImageJ software. (H) Light microscopic images of myofibers (left panels) and immunofluorescence staining of myogenin (red) and counterstained with DAPI (blue) in myofiber-derived MSCs (right panel) infected with either empty vector (upper panel) or miR-214 (lower panel) expressing lentivirus. (I) Myofiber-derived MSCs were assayed for myogenin expression and expressed as a percentage of total myofiber- derived MSCs analyzed (empty vector, blue bar, n=5, 1712 total cells; miR-214, red bar, n=3, 2318 total cells, * p<0.05). (J) Real-time PCR analysis of myogenin expression in myofiber-derived MSCs transduced with empty vector or miR-214. Data represents pooled total RNA from three independent infections and is presented as averaged replicates (**p<0.001). (K) Real time PCR analysis of miR-214 expression in myofiber-derived MSCs transduced with empty vector or miR-214. (L) Immunoblot of Ezh2, Su(fu), and tubulin in empty vector or miR-214-wt retroviral transduced C2C12 cells. (M) Immunoblot of Ezh2 and tubulin in MEFs transduced with empty vector, miR-214-wt, or mir-214-mut. 214-1 and 214-2 represent two different experiments conducted with miR-214-wt constructs. (N) Immunoblot of Ezh2 and tubulin in MEFs non-transfected, transfected with scrambled or anti-miR-214 oligomers. Bands corresponding to Ezh2 protein were quantified using the NIH Image J software. (O) Ezh2 mRNA expression in empty vector or miR-214-wt retrovirally-transduced MEFs.