miR-181a decreases cell proliferation and cell viability more potently in the ABC-like DLBCL compared with the GCB DLBCL cell lines. (A) Cell proliferation analyses. Cells were transfected with control or miR-181a plasmids, and at the noted time points post-transfection, 10⁵ cells were counted and labeled with [³H] thymidine (2 Ci/mL) for 4 hours. Cells were 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 per cell line. ABC-like DLBCL cell lines are noted in blue and GCB-like DLBCL cell lines in red. Statistical significance was determined by Student t test (****P < .0001) and the results are presented as mean ± SEM. (B) Anti–miR-181a cell proliferation rescue analyses. GCB-like (OCILY7 and OCILY19) and ABC-like (HBL1 and U2932) DLBCL cell lines were transfected with control or miR-181a plasmids. Twenty-four hours post-transfection, cells were counted and transfected again with either control or anti–miR-181a oligos. At 48 hours, cells were counted, labeled with [³H], and read as in panel A. Results are from 3 independent experiments in triplicates per cell line. Student t test (*P < .05) and the results are presented as mean ± SEM. (C) Cell viability FACS analyses in GCB-like (red) and ABC-like (blue) DLBCL cell lines. Three million cells per sample were transfected with control or miR-181a GFP plasmids. Four days post-transfection, the cells were stained with Annexin V–phycoerythrin (PE) and 7- aminoactinomycin D. The FACS analysis was performed on LSRFortessa-HTS, and the collected data were analyzed by FloJo software (Tree Star). The graphs depict cumulative results from 3 independent experiments showing the percent difference in live cells between the GFP⁺ miR-181a and GFP⁺ control transfected cells. Statistical significance was determined using two-way ANOVA (**P < .01) and the results are presented as mean ± SD. (D) Cell viability analyses in miR-181a inducible GCB-like and ABC-like DLBCL cell lines analyzed as in panel C. See also supplemental Figure 5.