CREL rescues the miR-181a–mediated cell death. (A) BCL2 cell proliferation rescue experiment. DOX-inducible U2932 cells were transfected with control empty vector or BCL2 plasmid lacking the 3′UTR (24 hours), and then induced to express control or miR-181a with DOX for 48 hours. At 72 hours post-transfection, 10⁵ cells were counted and labeled with [³H] thymidine (2 Ci/mL) for 4 hours. The cells were then transferred onto fiberglass filters and the radioactivity was read by a scintillation counter. Shown are the [³H] thymidine incorporation ratios of miR-181a to control from 3 independent experiments in triplicates. Statistical significance was determined by Student t test (***P < .001) and the results are presented as mean ± SEM. Shown below is a western blot control for BCL2 overexpression in U2932 transfected cells (48 hours). Densitometry analyses of the protein bands were normalized to the loading control β-actin (NIH software; ImageJ), and the relative band density values in miR-181a (+) or BCL2 samples were compared with the control (set as 1) and are noted below the blot. (B) BCL2 cell viability rescue analysis in inducible U2932 cells transfected as in panel A above. The cells were washed and stained with Annexin V–FITC and with an irreversible dead-cells labeling dye, allophycocyanin-Cy7, and gated on the PE Texas Red⁺ populations. The graph depicts cumulative results from 3 independent experiments, showing the percent difference in live cells between the induced miR-181a and the control cells. Statistical significance was determined by Student t test (***P < .001) and the results are presented as mean ± SD. (C) NF-κB transcription factors cell viability rescue analysis as in panel B above. Shown is a representative FACS analysis (i). The graph depicts cumulative results from 3 independent experiments (ii), and a western blot control for the protein overexpression in U2932 cells at 48 hours (iii). See also supplemental Figure 6. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, not significant; pcDNA, plasmid cytomegalovirus promoter DNA.