Journal: Molecular and Cellular Biology

Article Title: p38? Mitogen-Activated Protein Kinase Depletion and Repression of Signal Transduction to Translation Machinery by miR-124 and -128 in Neurons

doi: 10.1128/MCB.00695-12

Figure Lengend Snippet: p38α is posttranscriptionally repressed by miR-124 and -128. (A) Hek293 cells were transfected (+) with 0.1 μM pre-miR-124, pre-miR-128, or a control, scrambled pre-miRNA and lysates were probed for p38α, p38β, and tubulin expression. (Top) An immunoblot representative of four independent assays. (Bottom) The mean densitometric quantification of p38α expression levels in four separate experiments is shown below the immunoblot. Expression of p38α in control samples was normalized to tubulin and set at 100 arbitrary units; values are means plus standard deviations (error bars). (B) (Top) Schematic of firefly Luc reporters containing wild-type (wt) and mutant p38α 3′UTRs, as indicated. Hek293 cells were transfected with the indicated p38α reporter along with pcDNA5 miR-124, pcDNA5 miR-128, or pcDNA5 as a control. Firefly luciferase (Luc) expression was normalized to Renilla Luc levels (used as a transfection control). (Bottom) Unlabeled black bars indicate reporter expression from constructs containing wild-type, endogenous seed sequences in the p38α 3′UTR. Expression levels of reporters containing mutant seed sequences are indicated. pcDNA5-transfected control samples were set at 100 arbitrary units for each reporter. Values are the means plus SEM for four independent experiments. Values that are significantly different (P < 0.05) from the value for the control are shown by an asterisk.

Article Snippet: Cells were transfected with 0.1 μM pre-miR RNA hairpins (Ambion) or 0.1 μM small interfering RNA (siRNA) (Qiagen) and 15 μl Lipofectamine RNAiMax (Invitrogen) per well in 6-well plates for 18 h, then fresh medium was added, and the cells were allowed to recover for an additional 48 h. For immunoprecipitation (IP) assays, 0.1 μM siRNA was transfected into 15-cm dishes with 50 μl Lipofectamine RNAiMax for 18 h, and then fresh medium was added for an additional 48 h. Transfected Tet-inducible cells were serum starved in serum-free medium with doxycycline (1 μg/ml) for 18 h prior to treatment with inhibitors and harvesting.

Techniques: Transfection, Expressing, Western Blot, Mutagenesis, Luciferase, Construct

Journal: Journal of Extracellular Vesicles

Article Title: Identification of anti‐inflammatory vesicle‐like nanoparticles in honey

doi: 10.1002/jev2.12069

Figure Lengend Snippet: miR‐4057 in H‐VLNs was identified to suppress NLRP3 inflammasome activation. [a. Six most abundant miRNAs in H‐VLNs in Bowtie analysis. b. miR‐4057 inhibited inflammasome activation. 20 nM of miRNA mimic or miRNA (miR) negative control AllStars negative control siRNA were transfected. c. miR‐4057 dose‐dependently inhibited Casp1 autocleavage and IL1‐β secretion upon NLRP3 inflammasome activation. Different amounts of miRNA negative control were co‐transfected with miR‐4057 to ensure the total transfected RNAs of 20 nM. d. Inhibitor of miR‐4057 blunted the anti‐inflammasome activity of miR‐4057. 2 nM of miR‐4057 and different amounts of miRNA inhibitor and miR negative control were transfected to ensure the total RNAs of 20 nM. After 24 h, BMDMs were treated with LPS + ATP to activate the NLRP3 inflammasome. Data were presented as mean ± SEM. N = 3. * P < 0.05, ** P < 0.01 relative to macrophages treated with LPS+ATP (black bar). Tubulin showed equivalent loading of cell lysates].

Article Snippet: Briefly, 2 μl of Lipofectamine RNAiMAX were diluted in Opti‐MEM (ThermoFisher Scientific) and added to 20 nM of miRNA mimic (Qiagen) or AllStars negative control siRNA (Qiagen) in Opti‐MEM.

Techniques: Activation Assay, Negative Control, Transfection, Activity Assay