Journal: Carcinogenesis

Article Title: Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer

doi: 10.1093/carcin/bgw088

Figure Lengend Snippet: CPI-7c efficiently degrades MDM2 but leads to stabilization of p53 and induces global ubiquitination response. In ( A ), MCF-7 cells were treated with 5, 10, 15 and 20 µM CPI-7c and Nutlin-3, and expression levels of MDM2, ubiquitin and p53 were assessed by using immunoblotting and shown in the image. In panel ( B ), MCF-7 cells were grown in coverslips and treated with either vehicle or 10 and 20 µM of CPI-7c and Nutlin-3, respectively, for 24h and subjected to immunofluorescence staining and analyzed by confocal microscope. Merged confocal photographs represent the superimposition of green (p53) and red (MDM2) images, and the magnified area of the box was shown in inset pictures. Scale bar, 20 µm. Representative of three independent experiments. In panel ( C ), RT–PCR analysis shows the mRNA expression MDM2 and GAPDH genes in response to 10 and 20 µM doses of CPI-7c and Nutlin-3 treatment.

Article Snippet: Nutlin-3 and ChIP grade antibody for ubiquitin, and HDM2 (catalytic RING domain) peptide were purchased from Enzo Life Sciences.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Microscopy, Reverse Transcription Polymerase Chain Reaction

Journal: Carcinogenesis

Article Title: Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer

doi: 10.1093/carcin/bgw088

Figure Lengend Snippet: CPI-7c physically binds to RING and N-terminal domains of MDM2 and selectively induces ubiquitination of MDM2 as well as interferes at p53–MDM2 interaction. MCF-7 cells were treated with 10 µM dose of CPI-7c and Nutlin-3 for 24h, and protein lysates were prepared for immunoprecipitation using human anti-ubiquitin. Ubiqutinated MDM2 level was measured using western blotting of anti-MDM2 antibody with proper input control and shown in panel ( A ). Similarly, ( B ) represents the level of associated p53 when lysate was immunoprecipitated using anti-MDM2 antibody. Panel ( C ) represents standard scatchard exponential hyperbolic binding curve of direct physical binding of CPI-7c and Nutlin-3 with N-terminal peptide and RING domain peptide of MDM2 in left and right panels, respectively, along with respective dissociation equilibrium constant ( Kd ) values.

Article Snippet: Nutlin-3 and ChIP grade antibody for ubiquitin, and HDM2 (catalytic RING domain) peptide were purchased from Enzo Life Sciences.

Techniques: Immunoprecipitation, Western Blot, Binding Assay

Journal: PLoS ONE

Article Title: Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway

doi: 10.1371/journal.pone.0147509

Figure Lengend Snippet: Panel (A) shows the sequence of three partial non-canonical NF-κB RelB binding sites in the IDO1 promoter (bold nucleotides). The arrows indicate the primer sequences for detection of the three RelB binding sites. Panel (B) shows the PCR products after chromatin immunoprecipitation (ChIP). Immature human DCs were stimulated with the CTB-INS fusion protein vaccine for 0 (-) or 6 hr (+). Protein-DNA complexes were cross-linked, the DNA sheared and RelB genomic DNA complexes immunoprecipitated with RelB monoclonal antibody. After purification of the DNA, Real-time PCR was performed using primers flanking the three consensus NF-κB RelB binding sites in the human IDO1 promoter region shown in panel A. The Input control consists of PCR amplification of the IDO1 promoter obtained from total genomic DNA prior to immunoprecipitation. Lane M: DNA fragment size marker, Lanes 1, 2, 3: show the products of PCR amplification with primer sets detecting the three consensus RelB binding sites in the vaccinated human IDO1 promoter region (white arrows). Panel (C) Quantification of immunoprecipitation was performed for three experiments, by normalizing the intensity of each immunoprecipitated band to its input.

Article Snippet: Fragmented chromatin was immunopreciated with a ChIP-grade antibody against RelB (GeneTex, Irvine, CA, USA) which was coupled with Dynabeads ®, at 4°C overnight.

Techniques: Sequencing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, Control, Amplification, Marker

Journal: PLoS ONE

Article Title: Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway

doi: 10.1371/journal.pone.0147509

Figure Lengend Snippet: In the basal inactive state (left), the TRAF-cIAP complex catalyzes ubiquitination of NIK, leading to constitutive NIK degradation in the proteasome leaving p100-containing RelB complexes isolated in the cytoplasm. During activation (right), the TRAF-cIAP complex is recruited to the CD40 receptor. Upon ligand binding, TRAF2-mediated, ubiquitination of cIAP1/2 switches its ubiquitin ligase activity from NIK to TRAF3. The resultant TRAF3 degradation destabilizes the TRAF-cIAP complex allowing accumulation of newly synthesized NIK. Phosphorylated NIK then transfers a phosphate to IKKα. Now activated, IKKα phosphorylates p100 leading to its partial proteosomal degradation releasing p52: RelB heterodimers that translocate into the nucleus. Further, activated IKKα phosphorylates NIK, destabilizing it thereby limiting downstream activation events.

Article Snippet: Fragmented chromatin was immunopreciated with a ChIP-grade antibody against RelB (GeneTex, Irvine, CA, USA) which was coupled with Dynabeads ®, at 4°C overnight.

Techniques: Ubiquitin Proteomics, Isolation, Activation Assay, Ligand Binding Assay, Activity Assay, Synthesized

Journal: Scientific Reports

Article Title: Oestrogen Receptor-α binds the FOXP3 promoter and modulates regulatory T-cell function in human cervical cancer

doi: 10.1038/s41598-017-17102-w

Figure Lengend Snippet: Primary human T reg cells contain high levels of oestradiol hormone complexed with ERα. ( A ) Reverse-transcription PCR assessment of ERα and FOXP3 expression in T reg cells (TR), T effs (TE) and Naïve T cells (TN) isolated from cervical tumours (t) or blood (b) obtained from CxCa patients (p) or healthy male donors (d). The ERα -expressing breast cancer cell line MCF7 was included as a positive control. Data are representative of 20 independent experiments. Gels have been run under the same experimental conditions. Full-length gels are presented in Supplementary Figs. to . ( B ) E2 concentrations as quantified by ELISA in different T-cell subsets, MACS-purified and flow-sorted from tumour tissue of SCC cervix; T reg cells - TRt (CD4 + CD25 hi CD127 lo ), T effs − TEt (CD4+CD25 int ), cytotoxic T lymphocytes (CD3+CD8+cells) and peripheral blood naïve T cells – NT PB (CD4+CD25 − ). A total of 10 5 cells per subset were used for quantification. Graphs show mean ± SEM of n = 20 per subset (PB = peripheral blood; t = tumour). ( C ) Oestradiol complexes present in MACS/sort-purified tumour T reg cells as detected by immunoprecipitation of oestradiol complexes using anti-E2 antibodies followed by immunoblotting with anti-ERα antibodies (i) or isotype-matched control antibodies (ii). β-actin immunoblots were carried out using lysates pre-immunoprecipitated with anti β-actin antibodies. Data are representative of 6 independent experiments. Gels have been run under the same experimental conditions. Please see Figs. S4 and S5 for original blot pictures.

Article Snippet: ChIP on the fragmented DNA was performed using the low cell # ChIP kit (no. kch-maglow-016, Diagenode, Belgium) and ChIP-grade monoclonal antibodies against ERα (no.Mab-009-050, Diagenode) or a control Ig (provided in the kit).

Techniques: Expressing, Isolation, Positive Control, Enzyme-linked Immunosorbent Assay, Purification, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Oestrogen Receptor-α binds the FOXP3 promoter and modulates regulatory T-cell function in human cervical cancer

doi: 10.1038/s41598-017-17102-w

Figure Lengend Snippet: ERα control of FOXP3 expression in tumour-derived and peripheral blood T reg cells. ( A ) ERα and FOXP3 mRNA detection in CxCa T reg cells or MCF7 cells after treatment or not with the ERα antagonist ICI 182,780 (ICI; 1.8 µM) for 72 h in the presence or absence of exogenous oestradiol (E2;3.6 nM). Data are representative of 6 independent experiments. Gels have been run under the same experimental conditions. Original gel images are shown in Figs. S6–S8. ( B ) Relative expression levels of ERα and FOXP3 in peripheral blood T reg cells subjected to the same treatment as described in Fig. 3A (n = 6 healthy male donors). ( C ) Loss of ERα protein expression in CxCa T reg cells after 72 h treatment with ICI even in the presence of E2 supplementation throughout. Cellular extracts were immunoprecipitated using anti-ERα antibodies followed by immunoblotting with antibodies against ERα. The ERα-expressing breast cancer cell line MCF7 served as a positive control. β-actin immunoblots were carried out using lysates pre-immunoprecipitated with anti β-actin antibodies. Data are representative of 6 independent experiments. Gels have been run under the same experimental conditions. Please see Figs. S9 and S10 for original blot pictures (see also Fig. ). ( D ) FOXP3 protein expression in CxCa T reg cells after treatment or not with 1.8 µM ICI in the presence or absence of E2 for 72 h. Cell lysates were immunoprecipitated and immunoblotted using anti-FOXP3 antibodies. β-actin immunoblots were carried out using lysates pre-immunoprecipitated with anti β-actin antibodies. Data are representative of 6 independent experiments. Gels have been run under the same experimental conditions. Please see Figs.S12 and S13 for original blot pictures.

Article Snippet: ChIP on the fragmented DNA was performed using the low cell # ChIP kit (no. kch-maglow-016, Diagenode, Belgium) and ChIP-grade monoclonal antibodies against ERα (no.Mab-009-050, Diagenode) or a control Ig (provided in the kit).

Techniques: Expressing, Derivative Assay, Immunoprecipitation, Western Blot, Positive Control

Journal: Scientific Reports

Article Title: Oestrogen Receptor-α binds the FOXP3 promoter and modulates regulatory T-cell function in human cervical cancer

doi: 10.1038/s41598-017-17102-w

Figure Lengend Snippet: ERα-enriched regions of the FOXP3 locus. ChIP-qPCR analysis of the FOXP3 promoter and enhancer regions in chromatin fragments pulled down by anti-ERα antibodies (filled bars) or control IgG (open bars) in the cell lysates of blood T reg cells obtained from healthy male volunteers ( A and C ) or MCF7 cells ( B ). Data shown are mean ± SEM of 6 independent experiments performed in triplicate.

Article Snippet: ChIP on the fragmented DNA was performed using the low cell # ChIP kit (no. kch-maglow-016, Diagenode, Belgium) and ChIP-grade monoclonal antibodies against ERα (no.Mab-009-050, Diagenode) or a control Ig (provided in the kit).

Techniques:

Journal: Scientific Reports

Article Title: Oestrogen Receptor-α binds the FOXP3 promoter and modulates regulatory T-cell function in human cervical cancer

doi: 10.1038/s41598-017-17102-w

Figure Lengend Snippet: ERα protein interacts with FOXP3 protein in human tumour-derived T reg cells. CxCa T reg cells were treated or not with ICI in the presence or absence of exogenous E2 for 72 h before lysis. The cell lysates were then immunoprecipitated using anti-E2 antibodies and the protein complexes obtained were immunoblotted using antibodies against ERα and FOXP3. β-actin immunoblots were carried out using lysates pre-immunoprecipitated with anti β-actin antibodies. Data are representative of 6 independent experiments. Gels have been run under the same experimental conditions. Please see Figs. to for original blot pictures. See also Fig. .

Article Snippet: ChIP on the fragmented DNA was performed using the low cell # ChIP kit (no. kch-maglow-016, Diagenode, Belgium) and ChIP-grade monoclonal antibodies against ERα (no.Mab-009-050, Diagenode) or a control Ig (provided in the kit).

Techniques: Derivative Assay, Lysis, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: JMJD6 Regulates ERα Methylation on Arginine

doi: 10.1371/journal.pone.0087982

Figure Lengend Snippet: List of antibodies and their application.

Article Snippet: H4R3me2as (#39705) , Rabbit , Active motif , 1/1000 , 1/200.

Techniques: