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. 2015 Mar 24;35(8):1329–1340. doi: 10.1128/MCB.01446-14

FIG 2.

FIG 2

Excessive cell cycle arrest and apoptosis in miR-155−/− B cells. (A) Real-time RT-PCR quantification of the cyclin-dependent kinase inhibitor genes p15, p16, p21, and p57 was performed in miR-155 null or WT B cells following exposure to LPS–IL-4 (left panel) or NP-CGG immunization (right panel). We detected a significantly higher expression of all CDKi genes in miR-155−/− than in miR-155+/+ cells. Data in the left and right panels represent the mean ± SEM for nine and three independent quantifications (18 and 6 mice), respectively. Data are displayed as relative expression; all quantitative RT-PCRs were performed in triplicate. (B) Propidium iodide (PI) staining was used to determine the cell cycle profiles of mature B cells from three pairs of miR-155+/+ and miR-155−/− littermates exposed to LPS–IL-4 for 72 h. The absence of miR-155 was significantly associated with a higher percentage of G0/G1-arrested cells. (C) Annexin V staining was used to quantify apoptosis in three pairs of miR-155+/+ and miR-155−/− littermates exposed to LPS–IL-4 for 96 h. A significantly higher fraction of miR-155 null than WT cells were positive for annexin V. Data shown in panels B and C are mean ± SD of independent measurements performed in triplicate. (D) Mature B cells from two pairs of miR-155+/+ and miR-155−/− littermates were grown in medium containing LPS–IL-4 for 48 h and electroporated with control (ctrl) or p53-specific siRNA oligonucleotides, and the cell cycle profile was determined 24 h later. Knockdown of p53 abrogated the excessive G0/G1 arrest found in miR-155−/− B cells (compare si-ctrl versus si-p53-transfected cells). Western blots (bottom) confirmed the effective KD of p53 in this model. (E) Real-time RT-PCR of p15, p16, p21, and p57 in miR-155+/+ or miR-155−/− B cells following si-ctrl or si-p53 transfection. The higher levels of p15, p16, and p21 in miR-155−/− cells was significantly limited by the KD of p53. Data shown are the mean ± SD for the cells shown in panel D, displayed as relative expression normalized by the values obtained with miR-155+/+ cells. *, P < 0.05; **, P ≤ 0.01 (two-tailed Student t test).