Journal: Nature neuroscience

Article Title: Identification of a Unique TGF-? Dependent Molecular and Functional Signature in Microglia

doi: 10.1038/nn.3599

Figure Lengend Snippet: Identification of a miRNA microglia signature (a) Heatmap and hierarchical clustering of differentially expressed miRNAs in microglia, organ specific macrophages and immune cell populations based on 600 miRNA nCounter chip (see Source data – Figure 3). (b) miRNA transcript copies of highly expressed microRNAs in microglia and Ly6C monocyte subsets. Bars show mean normalized intensity ± s.e.m. of miRNA transcripts per 100 ng of total RNA ( n = 2). (c) qPCR validation of microglial miRNAs (miR-99a, miR-342-3p miR-125b-5p) and (d) inflammatory Ly6C + miRNAs (miR-15b, miR-148a, miR-223) in Ly6C monocytes, organ-specific macrophages and human fetal and adult microglia. Bars show mean normalized intensity ± s.e.m. ( n = 3). miRNA expression level was normalized against U6 miRNA using ΔCt. One representative of two individual experiments is shown.

Article Snippet: RNA isolation Total RNA was extracted using mirVanaTM miRNA isolation kit (Ambion) according to the manufacturer’s protocol.

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing

Journal: The EMBO Journal

Article Title: Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

doi: 10.15252/embj.201490546

Figure Lengend Snippet: SroC induces GcvB degradation by a base-pairing mechanism GcvB-SroC base-pairing regions. The compensatory base pair changes in GcvB and SroC are indicated in red. Salmonella Δ gcvB Δ sroC (JVS-5822) strain was cotransformed with pP L -GcvB and pBAD-SroC derivatives (see Supplementary Table S4). Each transformant was grown to OD 600 of 0.5 (0 min) and incubated for another 10 min in the presence of 0.2% l -arabinose. Total RNA was prepared from the cells and subjected to Northern blot analysis. The fold repression of GcvB was determined from three independent replicates; standard deviation is shown. The chromosomally modified wild-type sroC strain (JVS-10108), sroC ** mutant (JVS-10111) and sroC deletion mutant (JVS-5821) were grown to early stationary phase in LB medium (OD 600 of 2.0) prior to the addition of rifampicin. Cultures were harvested at the indicated time points after rifampicin treatment. RNA was isolated and analyzed by Northern blot as in Fig 3B . Source data are available online for this figure.

Article Snippet: At optical density (OD600 ) of 1.5, l -arabinose was added to cultures at a final concentration of 0.2% to induced SroC expression for 10 min, and total RNA was prepared with SV total RNA isolation system (Promega).

Techniques: Incubation, Northern Blot, Standard Deviation, Modification, Mutagenesis, Isolation

Journal: The EMBO Journal

Article Title: Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

doi: 10.15252/embj.201490546

Figure Lengend Snippet: Direct binding between GcvB and SroC sRNAs 20 nM of 5′-end-labeled GcvB and SroC were subjected to RNase T1 and lead(II) cleavage in the absence and presence of 100 nM cold sRNAs, SroC, and GcvB, respectively. Lane C: untreated RNA; lane T1: RNase T1 ladder of denatured RNA; lane OH: alkaline ladder. The position of G residues cleaved by RNase T1 is indicated at the left of picture. Secondary structures of GcvB and SroC, paired or alone. RNase T1 cleavage sites are indicated by arrowheads; color indicates those that appeared (red) or disappeared (blue) upon base-pairing. The base pairing sites BS1 and BS2 are shadowed. Source data are available online for this figure.

Article Snippet: At optical density (OD600 ) of 1.5, l -arabinose was added to cultures at a final concentration of 0.2% to induced SroC expression for 10 min, and total RNA was prepared with SV total RNA isolation system (Promega).

Techniques: Binding Assay, Labeling

Journal: The EMBO Journal

Article Title: Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

doi: 10.15252/embj.201490546

Figure Lengend Snippet: RNase E mediates SroC processing and GcvB degradation in distinct pathways Salmonella Δ gltIJKL rne + (JVS-9257) and Δ gltIJKL rne3071 (JVS-9258) strains transformed with pBAD- gltI or pBAD-SroC were grown to OD 600 of 0.3 at 28°C and further incubated at 44°C for 30 min (OD 600 of ˜0.5, indicated as time point 0). SroC expression was then induced for 10 min in the presence of 0.2% of l -arabinose. Total RNA was analyzed by Northern blots. 100 nM of 5′PPP or 5′P in vitro -transcribed preSroC was incubated with 100 nM of purified RNase E (1–529) at 30°C for the indicated time in the presence (lanes 10–17) or absence (lanes 2–9) of 100 nM Hfq. The same amount of preSroC was loaded in lane 1 as a control. SroC transcripts were detected using 5′-end-labeled oligonucleotide JVO-2907. Salmonella Δ gltIJKL rne + (JVS-9257) and Δ gltIJKL rneR169K (JVS-11001) strains transformed with pBAD- gltI or pBAD-SroC were grown to OD 600 of 0.5 (0 min) at 37°C and was further incubated for 10 min in the presence of 0.2% l -arabinose. The asterisk indicates transcriptional read-through to the rrnB terminator located downstream on the plasmid. Total RNA was prepared from the Salmonella strains and subjected to Northern blot analysis. 9S rRNA accumulated in the rneR169K strain. Estimated size from pUC8 marker is indicated on the right. Source data are available online for this figure.

Article Snippet: At optical density (OD600 ) of 1.5, l -arabinose was added to cultures at a final concentration of 0.2% to induced SroC expression for 10 min, and total RNA was prepared with SV total RNA isolation system (Promega).

Techniques: Transformation Assay, Incubation, Expressing, Northern Blot, In Vitro, Purification, Labeling, Plasmid Preparation, Marker

Journal: The EMBO Journal

Article Title: Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

doi: 10.15252/embj.201490546

Figure Lengend Snippet: Posttranscriptional regulation of GcvB by SroC Salmonella Δ sroC (JVS-5821), Δ gcvB Δ sroC (JVS-5822), and Δ sroC Δ hfq (JVS-9031) strains were transformed with pBAD-ctrl (pKP8-35) or pBAD-SroC (pYC6-4). Δ gcvB Δ sroC was cotransformed with pP L -GcvB (pMM03). Each strain was grown to OD 600 of 0.5 (0 min) and was further incubated for 10 min in the presence of 0.2% l -arabinose. Salmonella WT (JVS-1574), Δ gltIJKL (JVS-5823), ΔCA (JVS-10741), and Δ sroC (JVS-5821) strains were grown to OD 600 of 2.0 prior to the addition of rifampicin. Total RNA was analyzed by Northern blot to determine decay rates of GcvB. The half-lives were determined from three independent experiments; the standard deviation is indicated. Source data are available online for this figure.

Article Snippet: At optical density (OD600 ) of 1.5, l -arabinose was added to cultures at a final concentration of 0.2% to induced SroC expression for 10 min, and total RNA was prepared with SV total RNA isolation system (Promega).

Techniques: Transformation Assay, Incubation, Northern Blot, Standard Deviation

Journal: The EMBO Journal

Article Title: Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

doi: 10.15252/embj.201490546

Figure Lengend Snippet: SroC is the effector molecule for GcvB repression Expression changes of OppA and GcvB upon deletion of gltIJKL locus. WT (JVS-1574), Δ gltIJKL (JVS-5823), Δ gltJKL (JVS-10795), Δ sroC (JVS-5821), Δ gcvB Δ sroC (JVS-5822), and Δ gcvB (JVS-0236) strains were grown to early stationary phase (OD 600 of 2.0) in LB medium. Expression changes of OppA and GcvB by overexpression of gltI - sroC . Δ gltIJKL strain (JVS-5823) harboring pBAD-ctrl (lane 1), pBAD- gltI (pMM36) derivatives (lanes 2-5), or pBAD-SroC (lane 6) was grown to early stationary phase (OD 600 of 2.0) in LB medium supplemented with 0.02% l -arabinose. Data information: Upper two panels: Total protein was analyzed by Western blot to quantify OppA expression. GroEL served as a loading control. Lower three panels: Total RNA was analyzed by Northern blot, and expression of GcvB and SroC was monitored. 5S rRNA served as a loading control. Estimated size from pUC8 marker is indicated on the right. Source data are available online for this figure.

Article Snippet: At optical density (OD600 ) of 1.5, l -arabinose was added to cultures at a final concentration of 0.2% to induced SroC expression for 10 min, and total RNA was prepared with SV total RNA isolation system (Promega).

Techniques: Expressing, Over Expression, Western Blot, Northern Blot, Marker

Journal: American Journal of Translational Research

Article Title: Two-layer regulation of TRAF6 mediated by both TLR4/NF-kB signaling and miR-589-5p increases proinflammatory cytokines in the pathology of severe acute pancreatitis

doi:

Figure Lengend Snippet: AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for RNA isolation and qRT-PCR analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P

Article Snippet: Total RNA isolation and qRT-PCR analysisThe cultured cells and pancreatic tissues were subjected to total RNA isolation with a TRIzol reagent (Thermo Fisher Scientific, #15596026) following a method provided by the manufacturer.

Techniques: Transfection, Expressing, Isolation, Quantitative RT-PCR

Journal: American Journal of Translational Research

Article Title: Two-layer regulation of TRAF6 mediated by both TLR4/NF-kB signaling and miR-589-5p increases proinflammatory cytokines in the pathology of severe acute pancreatitis

doi:

Figure Lengend Snippet: Overexpression or downregulation of miR-589-5p affected the TLR4/NF-kB signaling. (A) Changes in miR-589-5p levels affected the protein levels of several members of the TLR4/NF-kB signaling pathway. Total cell extracts from MIA PaCa-2 cells transfected with miR-NC, miR-589-5p-mimic and anti-miR-589-5p were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) Changes in miR-589-5p levels did not affect NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were applied to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in the cytoplasmic fractions. The cytoplasmic fractions from MIA PaCa-2 cells transfected with miR-NC, miR-589-5p-mimic and anti-miR-589-5p were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions. The nuclear fractions from MIA PaCa-2 cells transfected with miR-NC, miR-589-5p-mimic and anti-miR-589-5p were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) Changes in miR-589-5p levels affected the expression of NF-kB targets. Cells used in (A) were used for RNA isolation and qRT-PCR analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P

Article Snippet: Total RNA isolation and qRT-PCR analysisThe cultured cells and pancreatic tissues were subjected to total RNA isolation with a TRIzol reagent (Thermo Fisher Scientific, #15596026) following a method provided by the manufacturer.

Techniques: Over Expression, Transfection, Expressing, Isolation, Quantitative RT-PCR