Figure 2

MyoD ~ E, RUNX1, and ZNF238 increase miR-206. (A) microRNA northern blots to detect the mature form of the indicated microRNAs in RD cells infected with either empty (control) retrovirus, or retrovirus expressing MyoD ~ E (MD ~ E). (B) RT-PCR using primers located in the pre- and pri-miR-206 sequence to detect the primary miR-206 transcript. TIMM17b is the internal control. (C) microRNA northerns as in 2A, in RD cells expressing a transcription factor as indicated. (D) microRNA northerns in C2C12 cells at various stages of differentiation ranging from undifferentiated myoblasts (50% GM), through beginning differentiation (90% GM) to myotubes (DM). (E) Immunostains for MHC in RD cells transiently transfected with either a pre-miR-206 RNA construct, or a negative control construct. Nuclei were stained with DAPI. (F) qPCR for CKM in RD cells treated as in E. (G) RD cells treated as in E were pulsed with BrdU for 24 h and then stained and counted to determine the extent of co-localization of MHC + myotubes and BrdU + nuclei. (H) RD cells transiently transfected as in E were pulsed for 24 h with BrdU-containing differentiation media before fixation and quantification of the percentage of BrdU positive cells. The percent reduction of BrdU + nuclei almost exactly equals the percent of cells found to be MHC + (not shown). (I) Luciferase activity in RD cells using a miR-206 promoter driven reporter and transiently transfected factors as indicated. ‘206 RUNX mutant’ indicates that the reporter has had a putative RUNX1 binding site mutated to prevent RUNX1 binding. Control indicates transfection with an empty plasmid. All luciferase assays were normalized to the results from a co-transfected renilla plasmid. (J) RUNX1 ChIP assays, both with normal PCR and qPCR results, at the miR-206 promoter and a control locus before (Control) and after (RUNX1) infection of the cells with empty or RUNX1-expressing retrovirus. All PCRs in the imaged gel (upper) were performed for the same number of cycles. qPCR results (lower) represent the mean ± SEM of two independent ChIP experiments. Relative enrichment is calculated as the ratio of the % of input amplified with antibody to the % of input amplified with no antibody. All other graphs in this figure represent the mean ± SEM of at least three independent experiments. * : P < 0.05; ** : P < 0.01; *** : P < 0.001.