Journal: Journal of Biological Chemistry

Article Title: MicroRNA-31 Sensitizes Human Breast Cells to Apoptosis by Direct Targeting of Protein Kinase C ϵ (PKCϵ)

doi: 10.1074/jbc.m112.414128

Figure Lengend Snippet: FIGURE 1. Overexpression of miR-31 reduces NF-B activity by a mechanism independent from the known direct targets RhoA and NF-B-inducing kinase (NIK; MAP3K14). a, NF-B activity in HEK293FT cells was analyzed by an NF-B reporter luciferase assay. Cells were co-transfected with the 3xKBL luciferase reporter plasmid, the pMir-Report--Gal reporter plasmid for normalization and the indicated MIRIDIAN miRNA mimics. 48 h after transfection, cells were stimulated using 20 ng/ml TNF- for 5 h, and relative luciferase activity was quantified (RLU, relative luciferase units). b, NF-B activity in HEK293FT cells was analyzed by Western blot stainingforactivephosphorylatedp65(Ser-536).CellsweretransfectedwiththeindicatedmiRNAmimics.48haftertransfection,cellswerestimulatedusing20ng/ml TNF- for 15 min, and protein lysates were prepared. GAPDH was used as loading control (ctrl). NF-B activity in MCF10A (c) and MDA-MB-231 (d) cells was analyzed byNF-Bp65nucleartranslocationassay.CellsweretransfectedwiththeindicatedmiRNAmimics.48haftertransfection,cellswerestimulatedusing20ng/mlTNF- for 15 min and immunostained for p65. Nuclei were stained with DAPI, and the ratio of “active” cells with mainly nuclear localization of p65 over “passive” cells with mainlycytoplasmiclocalizationofp65wasdeterminedusinganOlympusScanRhigh-contentscreeningmicroscope.e,expressionlevelsofmiR-31upontransfection with miRNA mimics were determined by qRT-PCR. f, NF-B activity in HEK293FT cells was analyzed by NF-B reporter luciferase assay. Cells were co-transfected with the3xKBLluciferasereporterplasmid,thepMir-Report--GalreporterplasmidfornormalizationandtheindicatedsiRNAs.48haftertransfection,cellswerestimulated using 20 ng/ml TNF- for 5 h, and relative luciferase activity was quantified.

Article Snippet: The following antibodies were used: rabbit monoclonal phosphoNF- B p65 (Ser-536), rabbit anti-PKC (22B10) (both Cell Signaling Technology, Danvers, MA), rabbit anti-actin (20-33) (Sigma-Aldrich).

Techniques: Over Expression, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Western Blot, Control, Staining, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: MicroRNA-31 Sensitizes Human Breast Cells to Apoptosis by Direct Targeting of Protein Kinase C ϵ (PKCϵ)

doi: 10.1074/jbc.m112.414128

Figure Lengend Snippet: FIGURE 3. Knockdown of PRKCE reduces NF-B activity. a, NF-B activity in HEK293FT cells was analyzed by an NF-B reporter luciferase assay. Cells were co-transfected with the 3xKBL luciferase reporter plasmid, the pMir-Report- -Gal reporter plasmid for normalization and the indicated siRNAs. 48 h after transfection, cells were stimulated using 20 ng/ml TNF- for 5 h, and relative luciferase activity was quantified. NF-B activity in MCF10A (b) and MDA-MB- 231 (c) cells was analyzed by NF-B p65 nuclear translocation assay. Cells were transfected with the indicated miRNA mimics. 48 h after transfection, cells were stimulated using 20 ng/ml TNF- for 15 min and immunostained for p65. Nuclei were stained with DAPI and the ratio of active cells with mainly nuclear localization of p65 over passive cells with mainly cytoplasmic local- ization of p65 was determined using an Olympus ScanR high-content screen- ing microscope. d, NF-B activity in HEK293FT, MCF10A, and MDA-MB-231 cells was analyzed by Western blot staining for active phosphorylated p65 (Ser-536). Cells were transfected with the indicated miRNA mimics. 48 h after transfection, cells were stimulated using 20 ng/ml TNF- for 15 min, and pro- tein lysates were prepared. GAPDH was used as loading control.

Article Snippet: The following antibodies were used: rabbit monoclonal phosphoNF- B p65 (Ser-536), rabbit anti-PKC (22B10) (both Cell Signaling Technology, Danvers, MA), rabbit anti-actin (20-33) (Sigma-Aldrich).

Techniques: Knockdown, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Nuclear Translocation Assay, Staining, Microscopy, Western Blot, Control

Journal: Immunity

Article Title: C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

doi: 10.1016/j.immuni.2007.03.012

Figure Lengend Snippet: Figure 5. DC-SIGN-Mediated Raf-1-Dependent Signaling Induces p65 Phosphorylation on Ser276 and Acetylation, after Concur- rent TLR Signaling (A) DC-SIGN signaling does not influence nuclear translocation of different NF-kB subunits. DNA-binding ELISA of the NF-kB subunits p65, RelB, c-Rel, p50, and p52 in nuclear extracts of human iDCs treated as indicated for 1 hr. NF-kB was allowed to bind to oligonucleotides containing the NF-kB-binding consensus sequence; specific antibodies were used to detect the different subunits within the bound complexes. Results are pre- sented as the means ± SD from two independent experiments. (B and C) DC-SIGN signaling induces p65 phosphorylation on Ser276 through Raf-1. ELISA of phospho-Ser536-p65 (B) or phospho-Ser276-p65 (C) in cell lysates of human iDCs treated as indicated for 30 min; cells were preincubated for 2 hr with Raf inhibitor GW5074 as indicated. Results are pre- sented as the means ± SD from three independent experiments. (D) DC-SIGN induces acetylation of p65. ELISA with acetyl-lysine antibodies on captured p65 from cell lysates of human iDCs treated as indicated for 30 min; cells were preincubated with Raf inhibitor GW5074 as in (C). Results are presented as the means ± SD from three independent experiments. (E) Acetylation of p65 is essential to ManLAM-induced IL-10 upregulation. Relative IL-10 mRNA production by human iDCs treated with different ligands for 6 hr as indicated; cells were preincubated for 2 hr with anacardic acid (AA), an inhibitor of the histone acetyltransferases p300 and CBP. IL-10 mRNA was determined as described in Figure 1A. Results are presented as the means ± SD from three independent experiments.

Article Snippet: DNA Binding, Acetylation, and Phosphorylation of NF-kB DC nuclear extracts were prepared with the NucBuster protein extraction kit (Novagen, Madison, WI), and 5 mg of nuclear extract was used to determine the specific subunits within the DNA binding NF-kB dimers with the TransAM NF-kB family kit (Active Motif, Carlsbad, CA), according to the manufacturer’s protocol. p65 from cell lysates was captured with the Pathscan phosphoNF-kB p65 (Ser536) sandwich ELISA kit (Cell Signaling), according to the manufacturer’s protocol.

Techniques: Phospho-proteomics, Translocation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: Immunity

Article Title: C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

doi: 10.1016/j.immuni.2007.03.012

Figure Lengend Snippet: Figure 6. DC-SIGN-Induced Acetylation Prolongs Nuclear p65 DNA Binding and Enhances the IL-10 Transcription Rate (A) DC-SIGN signaling prolongs nuclear p65 DNA binding compared to LPS signaling alone. DNA binding was determined as described in Figure 5A. Results are presented as the means ± SD from two independent experiments. (B) Acetylation of p65 prolongs nuclear p65 DNA binding. ELISA of p65 in nuclear extracts of hu- man iDCs treated similar as in (A); cells were pre- incubated with Raf inhibitor GW5074 and HAT inhibitor anacardic acid (AA) as in Figures 5C and 5E. DNA binding was determined as in (A). Results are presented as the means ± SD from two independent experiments. (C) DC-SIGN signaling enhances the IL10 tran- scription rate. Relative IL-10 transcription rate in nuclei isolated from human iDCs treated sim- ilar as in (A); cells were preincubated for 2 hr with the blocking DC-SIGN antibody AZN-D2 as indi- cated. Transcription by isolated nuclei was initi- ated in the presence of 16-biotin-UTP; after 20 min of transcription, nuclei were lysed, RNA with incorporated biotin was isolated, and quan- titative real-time PCR analysis was performed to determine the relative IL-10 RNA production. The amount of RNA produced in 20 min is repre- sentative of the transcription rate. The IL-10 RNA production by LPS-stimulated cells was set at 1. Results are presented as the means ± SD from two independent experiments.

Article Snippet: DNA Binding, Acetylation, and Phosphorylation of NF-kB DC nuclear extracts were prepared with the NucBuster protein extraction kit (Novagen, Madison, WI), and 5 mg of nuclear extract was used to determine the specific subunits within the DNA binding NF-kB dimers with the TransAM NF-kB family kit (Active Motif, Carlsbad, CA), according to the manufacturer’s protocol. p65 from cell lysates was captured with the Pathscan phosphoNF-kB p65 (Ser536) sandwich ELISA kit (Cell Signaling), according to the manufacturer’s protocol.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Isolation, Blocking Assay, Real-time Polymerase Chain Reaction, Produced