HeLa S3 cells (6 x 104 cells/well) were transfected with a negative control or an miR-1 Mimic using HiPerFect Transfection Reagent. Twenty-four hours prior to measurement, cells were transfected with a luciferase reporter construct carrying an miR-1 binding site using Attractene Transfection Reagent. After the time points indicated, miR-1 levels were detected by measuring luciferase activity.
HDAC4 is predicted to be a target of miR-1 and has 2 miR-1 binding sites in the 3' UTR (Chen, J.F. et al.  The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat. Genet. 38, 228.). HeLa S3 cells (8 x 104 cells/well) were transfected using HiPerFect Transfection Reagent and a range of concentrations of miR-1 Mimic. miR-1 #1, miR-1 #2, and miR-1 #3 are different batches of miR-1 Mimic. After 24 and 48 hours, [A] HDAC4 protein level was analyzed by western blotting. Tubulin was also analyzed as an internal control (not shown). No effect was observed after 24 hours. After 48 hours, protein level was significantly decreased. [B] HDAC4 transcript level was analyzed by real-time PCR using the miScript System after transfection using 50 nM miR-1 Mimic. The negative control was AllStars Negative Control. Results were normalized to GAPDH and to the negative control and are presented as 2-ΔΔCT. Transcript levels did not change significantly at either the 24-hour or 48-hour time point.
miR-1 is not endogenously expressed in HeLa S3 cells. miR-16 is endogenously expressed at high levels in HeLa S3 cells (Lagos-Quintana, M. et al.  Identification of novel genes coding for small expressed RNAs. Science 294, 853.). Using HiPerFect Transfection Reagent, HeLa S3 cells (6 x 104 cells/well) were transfected with a luciferase reporter construct with a binding site for miR-1 in the 3' UTR; cotransfected with a luciferase reporter construct with a binding site for miR-1 in the 3' UTR and with a miR-1 Mimic; or transfected with a luciferase reporter construct with a binding site for miR-16 in the 3' UTR. Twenty-four hours later, luciferase activity was measured. miR-1 Mimic resulted in a similar decrease in luciferase level as endogenous miR-16.