Figure 6. miRNA-specific regulation of cell excitability and target protein abundance.

(A) Cultured cortical neurons were transfected with expression constructs for pre-miR-132, pre-miR-219 (not shown) or pre-miR-1 (not shown), along with a DsRed.T3-NLS-PEST expression plasmid as a transfection marker (transfection ratio 3:1 of pre-miR construct: DsRed). Negative controls were cultures transfected with empty vector (pcDNA3.1), and adjacent untransfected neurons (DsRed-negative); internal Ca²⁺ levels were monitored using time-lapse digital microscopy of Fluo-4. Representative data from Fluo-4 loaded (green) untransfected and transfected (red) neurons stimulated with K⁺ (20 mM), glutamate (20 μM) and NMDA (20 μM). (B) Peak ± SEM Ca²⁺ responses of untransfected and transfected neurons. Data for each experiment were normalized to the peak response of untransfected neurons, which was set equal to a value of 1. * p<0.01. (C) HEK 293 cells were transfected with expression constructs for SCOP or FLAG-RFX4, along with pre-miR-132, pre-miR-219 or pre-miR-1 and DsRed.T3-NLS-PEST (transfection ratio 2:6:1 of target: pre-miR construct: DsRed). Forty-eight hours after transfection, cells were fixed and immunolabelled for SCOP or FLAG-RFX4. Representative panels show target expression when transfected with each pre-miRNA. Note the reduced expression of SCOP when cotransfected with pre-miR-219, and the reduced expression of FLAG-RFX4 when cotransfected with pre-miR-132. (D) Mean immunolabeling intensity for SCOP and FLAG-RFX4 under the three cotransfection conditions. Y-axis denotes fluorescent intensity units (0-4096 scale). Values are presented as mean ± SEM. n=200–250 cells per group. *p<0.05 (two-tailed Student’s t-test).