miR-150 regulates FOXP1 levels in FL/tFL. (Ai) Statistical analysis of the effect of synthetic miR-150 on FOXP1 levels in WSU-NHL cells (48 hours; n = 3). WSU-NHL cells were transfected with siRNA against FOXP1 (siFOXP1) or siRNA negative control (siNeg. Ctrl) or miR-150 mimic (miR-150 MIMIC) or miRNA negative control (miR Neg. Ctrl) and harvested after 48 hours. The siRNA against FOXP1 serves as a positive control. The blot images were quantified with UVItec Alliance 4.7, and the FOXP1/β-actin ratio in the first sample (siNeg. Ctrl) was arbitrarily set at 1. The differences were tested by paired t test. (Aii) Representative immunoblot of FOXP1 levels in WSU-NHL cells transfected with siRNA against FOXP1 or miR-150 mimic as described in panel Ai. *Two bands identified by the anti-FOXP1 antibody represent two isoforms of FOXP1 protein. (B) The luciferase activity in HEK293T cells cotransfected with miR-150 mimic (400 nM) and luciferase reporter construct (15 ng), containing the 3′UTR region of wild-type or mutated FOXP1 (supplemental Methods). As a negative control, we used a control miRNA mimic (miR Neg. Ctrl; 400 nM). Luciferase activity was measured 24 hours posttransfection and compared by paired t test. (C) Statistical analysis of FOXP1 staining in paired FL-tFL samples from all 14 patients with available material (patient characteristics listed in supplemental Table 1 [FL patients 5-10 and 12-19]). Percentages of samples with low (≤30%), intermediate (inter; 30%-70%), and high (≥70%) percentages of FOXP1⁺ cells are shown. The number in each segment of the column indicates the number of samples with the corresponding intensity of IHC staining. Each arrow indicates the change in the IHC staining category in each of the analyzed pairs (note that the angle of the line does not show the trend of increase/decrease in percentage of positive cells). The statistical differences were tested by Wilcoxon matched-pairs test. Representative images of the scoring of FOXP1 staining in FL are shown in panel D and in supplemental Figure 14. (D) Representative examples of FOXP1 IHC staining between paired samples of FL (upper) and tFL (lower). Original magnification ×400. (E) Differences in miR-150 expression levels between FL samples with low (≤30%), intermediate (30%-70%), and high (≥70%) percentages of FOXP1⁺ cells from 30 FL patients with available material (FOXP1 staining performed as tissue microarray blocks; supplemental Methods). P values were assessed by unpaired t test. Representative images of the scoring of FOXP1 staining in FL are shown in supplemental Figure 14. (F) Cell-cycle analysis of WSU-NHL cells transfected with synthetic miR-150 mimic or siRNA against FOXP1 or negative controls for miRNA or siRNA. Cells were harvested after 24 hours (n = 10 independent experiments) and analyzed for cell cycle (supplemental Methods; supplemental Figure 16). P values were assessed by paired t test. (G) The Ki67 proliferation index in FL samples with low (≤30%), intermediate (30%-70%), or high (≥70%) percentage of FOXP1⁺ cells from 27 FL patients with available material (FOXP1 staining performed as tissue microarray blocks; supplemental Methods). P values were assessed by unpaired t test. NS, not significant.