miR-107 expression in response to LPS over time in primary and MyD88- and p65-deficient cells. a, primary BMDM (upper) and PBMC (lower) were stimulated with LPS (100 ng/ml) for the times indicated. Expression of miR-107 was measured by RT-PCR. Results were normalized to the endogenous control RNU6b, a ribosomal RNA and are represented as -fold stimulation over the unstimulated control. Results in all cases are shown as relative expression (R.E.). b, primary BMDM (upper) and PBMC (lower) were stimulated with LPS (100 ng/ml) for the times indicated. Expression of miR-146a was measured by RT-PCR in the same manner as for miR-107. c, primary BMDM were stimulated with LPS (100 ng/ml) for the times indicated, and pri-miR-107 (left) and pre-miR-107 (right) were measured by RT-PCR. Results were normalized to the endogenous control GAPDH and are represented as -fold stimulation over the nonstimulated control. d, left, WT I-BMDM, MyD88-deficient I-BMDM, and Trif-deficient I-BMDM were stimulated with LPS (100 ng/ml) for 8 or 24 h. Right, WT MEF or p65-deficient MEF were stimulated with LPS (100 ng/ml) for the times indicated. The horizontal axis in all graphs represents time in hours of stimulation. In all cases, results are expressed as mean ± S.E. (error bars) of triplicate determinations from three separate experiments. *, p < 0.05;**, p < 0.01; ***, p < 0.001.

CDK6, Dicer1, and HIF-1α expression in response to LPS over time and the effect of miR-107 levels on CDK6. a, upper, primary BMDM were stimulated with LPS (100 ng/ml) for the times indicated. Expression of CDK6 was measured by RT-PCR in the same manner as for PanK1α. R.E., relative expression. Lower, cells were also assessed for CDK6 and β-actin protein expression. Relative intensity (R.I.) is shown below. b, upper, WT I-BMDM, MyD88-deficient I-BMDM, and Trif-deficient I-BMDM were stimulated with LPS (100 ng/ml) for 8 or 24 h and analyzed for CDK6 expression by RT-PCR. Lower, WT MEF or p65-deficient MEF were stimulated with LPS (100 ng/ml) for the times indicated. c, I-BMDM were stimulated with LPS (100 ng/ml) for the times indicated. Expression of Dicer1 (left) and HIF-1α (right) was measured by RT-PCR. In all cases, results are expressed as mean ± S.E. (error bars) of triplicate determinations from three separate experiments. *, p < 0.05;**, p < 0.01.

Effect of CDK6 expression on adhesion of macrophages in response to LPS and the effect of LPS on CDK6 knock-out mice. a, adhesion of WT and CDK6-deficient BMDM in response to LPS was assessed using the xCELLigence system. 5 × 10⁵ CDK6-deficient BMDM or wild-type BMDM were plated in the E-plate and stimulated with LPS (100 ng/ml). b, wild-type (WT) control mice and CDK6-deficient (CDK6^−/−) mice 8 weeks of age were injected intraperitoneally with LPS (30 mg/ml) and monitored over a period of 5 days; results are plotted as a percentage of total numbers (n = 14 mice/group). Kaplan-Meier analysis demonstrated a significant difference in survival between CDK6^−/− mice and WT (log-rank test Cdk6^−/− versus WT; p = 0.01). c, ELISA of mouse TNF-α in blood samples from wild-type and CDK6-deficient mice (n = 14 mice/group), 1 h and 4 h after LPS injection is shown. **, p < 0.01; ***, p < 0.001. d, schematic illustrates the possible role of miR-107 in TLR4 signaling and on macrophage cell adhesion in response to LPS. In response to LPS, MyD88 adaptor molecules initiate a signaling cascade, which leads to the activation of the NF-κB subunit p65 and the repression of PPAR-α. This repression leads to the decrease in PanK1α expression and the subsequent decrease in miR-107 because of its intronic location in PanK1α. This decrease in miR-107 releases its hold on CDK6, leading to an increase in its expression and an increase in macrophage cell adhesion.

PanK1α and PPAR-α expression in response to LPS over time. a, upper, primary BMDM were stimulated with LPS (100 ng/ml) for the times indicated. Expression of PanK1α was measured by RT-PCR. Results were normalized to the endogenous control GAPDH and are represented as -fold stimulation over the nonstimulated control. R.E., relative expression. Lower, primary BMDM were stimulated with LPS (100 ng/ml) for the times indicated and assessed for PanK1α and β-actin. Relative intensity (R.I.) is shown to compare intensity of the Western blot bands relative to time 0. b, upper, WT I-BMDM, MyD88-deficient I-BMDM, and Trif-deficient I-BMDM were stimulated with LPS (100 ng/ml) for 8 or 24 h. Lower, WT MEF or p65-deficient MEF were stimulated with LPS (100 ng/ml) for the times indicated. c, upper, primary BMDM were stimulated with LPS (100 ng/ml) for the times indicated. Expression of PPAR-α was measured by RT-PCR in the same manner as PanK1α. Lower, primary BMDM were stimulated with LPS (100 ng/ml) for the times indicated and assessed for PPAR-α and β-actin. Relative intensity is shown below. d, upper, WT I-BMDM, MyD88-deficient I-BMDM, and Trif-deficient I-BMDM were stimulated with LPS (100 ng/ml) for 8 or 24 h. Lower, WT MEF or p65-deficient MEF were stimulated with LPS (100 ng/ml) for the times indicated. e, I-BMDM were pretreated with 10 μm WY14643 for 30 min before the addition of LPS (100 ng/ml) for 24 h. Expression of miR-107, PanK1α, and PPAR-α was measured by RT-PCR. Results were normalized to the endogenous controls RNU6b (miR-107) or GAPDH (PanK1α and PPAR-α) and are represented as -fold stimulation over the dimethyl sulfoxide (DMSO) control. f, ELISA of mouse TNF-α was measured in supernatants of samples pretreated with WY14643 before LPS stimulation. In all cases, results are expressed as mean ± S.E. (error bars) of triplicate determinations from three separate experiments. *, p < 0.05;**, p < 0.01.

Effect of miR-107 expression on adhesion of macrophages in response to LPS. a, left, I-BMDM were transfected with 50 nm antisense miRNA oligonucleotides and stimulated with LPS (100 ng/ml) for the times indicated. Expression of CDK6 was measured as before using RT-PCR. R.E., relative expression. Right, cells were also assessed for CDK6 and β-actin protein expression in response to LPS for the times indicated. Relative intensity (R.I.) is shown below. b, left, I-BMDM were transfected with 50 nm precursor miRNA oligonucleotides and stimulated with LPS (100 ng/ml) for the times indicated. Expression of CDK6 was measured as before. Right, cells were also assessed for CDK6 and β-actin protein expression in response to LPS for the times indicated. Relative intensity is shown below. c, CDK6 3′-UTR luciferase plasmid and CDK6 mutant 3′-UTR luciferase plasmid were co-transfected with 50 nm anti-miR-107, anti-miR-control, pre-miR-107, or pre-miR-control in HEK293T cells. Luciferase activity was measured, and results were normalized for TK-Renilla activity. Data are shown as percent luciferase relative to control. Results are expressed as mean ± S.E. (error bars) of triplicate determinations from three separate experiments. d, upper, adhesion of macrophages transfected with anti-miR-107 or pre-miR-107 was assessed in response to LPS. 5 × 10⁵ Raw264.7 cells were transfected with 50 nm anti-miR-107 or anti-miR-control and stimulated with LPS (100 ng/ml) for the times indicated and assessed for adhesion in real time using the xCELLigence system. Lower, 5 × 10⁵ Raw264.7 cells were transfected with 50 nm pre-miR-107 or pre-miR-control and stimulated with LPS (100 ng/ml) and assessed for adhesion in real time using the xCELLigence system. Data are plotted as -fold change cell index or the -fold change in cell index values between LPS-stimulated cells and the untreated control cells. e, I-BMDM were transfected with 50 nm antisense miRNA oligonucleotides and stimulated with LPS (100 ng/ml) for 24 h. ELISA of mouse TNF-α was measured. In all cases, results are expressed as mean ± S.E. (error bars) of quadruplicate determinations from three separate experiments. *, p < 0.05;**, p < 0.01; ***, p < 0.001.