Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: Identification of YY1 as a potential transcription regulator for FEN1. A . Top 10 hits of the transcription factors (TFs) that were predicted by TF Research Web sites: Match1.0-public, PROMO, and TFSEARCH. B . The oligo probes were designed to cover different regions of the predicted FEN1 promoter. Probes a, b, and c correspond to the region −290 to −230, −150 to −90, and −60 to 0, respectively. C . The silver staining image of oligo-pulled-down assays using HeLa cell extracts. b SNP : probe b with three SNP sites, r: a probe with random DNA sequences. The unique band, which is indicated by a box, was subjected to MS analysis. D . Top 10 hits of the MS analysis of the unique protein band as specified in Panel C .

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Silver Staining

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: YY1 binds to the conserved YY1 binding motif in the FEN1 promoter region. A . Sequence alignment of the conserved YY1 binding motif in different proteins. B . EMSA analysis of YY1 binding to the YY1 binding motif in the FEN1 promoter. Recombinant YY1 was incubated with different biotin-labeled DNA probes. The sequences of the Probe N, Probe P, WT FEN1and MUT FEN1 can be found in Additional file : Table S6. The free probe and YY1/DNA complex were resolved in 5% native PAGE. C . EMSA assay on YY1 and FEN1 oligo in the presence of non-specific IgG or the anti-YY1 antibody. D . ChIP analysis of YY1 binding to the FEN1 promoter region. Specific YY1-bound DNA in MCF7 cell extracts was pulled down by an anti-YY1 antibody. The YY1-bound FEN1 sequence was amplified by PCR. The sequence for the FEN1 promoter specific primer can be found in the Additional file : Table S6 as FEN1 (YY1). The PCR product was analyzed by 1% agarose electrophoresis.

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Binding Assay, Sequencing, Recombinant, Incubation, Labeling, Clear Native PAGE, Amplification, Electrophoresis

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: Overexpression of YY1 inhibits FEN1 promoter-driven protein expression. A . YY1 was overexpressed in 293 T cells and its impact on the FEN1 protein level was evaluated by western blot using the anti-Flag or anti-FEN1 antibody. B . The pCMV-Flag-YY1 expression vector and the pGL4.0-FEN1 promoter-EGFP vector, or pGL4.0 EGFP vector was co-transfected into 293 T cells. The EGFP expression was detected by semi-quantitative PCR (Upper panel) and quantitative PCR (lower panel). C . The overexpression of Flag-tagged YY1 was confirmed by western blot using the anti-Flag antibody. D and E . EGFP protein levels with or without YY1 overexpression was measured by FACS. Panel D shows the representative FACS images. Panel E is the quantification of FACS. Values are means ± s.d. of four independent experiments. p value was calculated by the two-tail student’s t-test. F . Knockdown of YY1 in 293 T (Left Panel) and MCF7 (right panel) cells. The YY1 and FEN1 expression was measured by quantitative PCR. The mRNA level was normalized with corresponding mRNA level of GADPH, and the normalized mRNA level of YY1 or FEN1 in the cells treated with control siRNA was arbitrarily set as 1. Values are means ± s.d. of three independent experiments. p value was calculated by the two-tail student’s t-test.

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Over Expression, Expressing, Western Blot, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Knockdown, Control

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: DNA damaging agents MMC and Taxol inhibit YY1 expression but induce FEN1 expression. A . and B . YY1 and FEN1 expression in MDA-MB-231 breast cancer cell line in response to the MMC and Taxol treatment. The mRNA level (A) and protein level (B) were measured by quantitative PCR and Western blot. The left panel in B showed the quantification of Western blot results. All experiments were independent carried out at least three times. C . Analysis of YY1 binding to the FEN1 promoter in response to the MMC treatment. Cells were treated with MMC, and the level of YY1-bound FEN1 promoter was evaluated by the ChIP assay. The lowest DNA staining density is arbitrarily set as 1. D . western blot confirmed the overexpression of Flag-tagged YY1 in 293 T cells. The β-actin(ACTB) was used as control. E . and F . The survivorship of 293 T cell and 293 T cell harboring a YY1 expression plasmid, pCMV-Flag-YY1, or the empty vector, under treatment of mytomycine C (MMC) (Panel E) or taxol (Panel F) . In both panels, the cells were treated with indicated concentrations of MMC or taxol for 48 hours. The number of survival cells was counted. The survival rate of the untreated cells with or without YY1 overexpression was arbitrarily set as 1.

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Staining, Over Expression, Control, Plasmid Preparation

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: FEN1 gene expression in response to chemotherapeutic drug and DNA damage agent treatments. A . Macro-image of hybridization of the 32 -P-labeled FEN1 ORF DNA fragment with the expression array, which contains cDNA from different cells lines treated with different DNA-damaging agents. In the control hybridization, 32 -P-labeled ubiquitin ORF DNA fragment was used . B . The relative fold changes of the density of the hybridized spots. All FEN1 hybridization signals were normalized with corresponding ubiquitin signals. In each cell line, the normalized signal in the untreated control sample was arbitrarily set as 1, and the fold change was calculated by comparing the normalized signal in a specific sample to that of the untreated control sample.

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Gene Expression, Hybridization, Labeling, Expressing, Control, Ubiquitin Proteomics

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: Associations between FEN1, YY1 protein, or their combination and OS or DFS. A . FEN1 Kaplan Meier survival plot with breast cancer patient cohort in the Ivshina data base. The black line indicates FEN1 high expression while the red line indicates FEN1 low expression (‘high’ and ‘low’ determined by median expression). Patients with FEN1 high expression: 132; patients with FEN1 low expression: 117, Log-rank p = 0.0007. B . Low expression of YY1 and high expression of FEN1 and OS in the CMU cohort. The black line indicates YY1 low but FEN1 high expression and red line indicates other types. Patients with YY1 low but FEN1 high expression: 154; patients with other types: 113, Log-rank p = 0.027. C . Low expression of YY1 and high expression of FEN1 and DFS in the CMU cohort. The black line indicates YY1 low but FEN1 high expression and red line indicates other types. Patients with YY1 low but FEN1 high expression: 154; patients with other type: 113, Log-rank p = 0.048.

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Expressing

Journal: Nature Communications

Article Title: HLA-DP 84Gly constitutively presents endogenous peptides generated by the class I antigen processing pathway

doi: 10.1038/ncomms15244

Figure Lengend Snippet: ( a ) K562 aAPCs were transiently transfected with the indicated combinations of genes and cultured in the presence or absence of 0.02 μM bortezomib or 0.02 μM carfilzomib for 48 h. Total cell lysates were immunoblotted with anti-MAGE-A3, anti-HLA-class I or anti-β-actin mAb. ( b , c ) K562/DP4/Ii cells were transiently transfected with a retrovirus vector encoding IRES-EGFP (control), or a native ( b ) or endosome-targeted ( c ) form of MAGE-A3 linked with IRES-EGFP. Cells were then cultured with carfilzomib at the indicated concentrations for 48 h. Transient transfection efficiencies were normalized to EGFP expression measured by flow cytometry. DP4/MAGE-A3 243–258 CD4 + T cells were stimulated with the K562/DP4/Ii transfectants and IFN-γ secretion was measured by ELISPOT analysis. Data shown represent means±s.d.'s of triplicates. ( d ) DP4/MAGE-A3 243–258 CD4 + T cells were stimulated with the indicated K562-based aAPCs pulsed with tetanus toxin 947–967 (control) or MAGE-A3 243–258 peptide, and IFN-γ secretion was evaluated by ELISPOT assays. Data shown represent means±s.d.'s of triplicates. ( e , f ) The indicated K562-based aAPCs were transiently transfected with a retrovirus vector encoding IRES-EGFP (control) or a native ( e ) or endosome-targeted ( f ) form of MAGE-A3 linked with IRES-EGFP. Transient transfection efficiencies were normalized to EGFP expression measured by flow cytometry. DP4/MAGE-A3 243–258 CD4 + T cells were stimulated with the indicated aAPCs and IFN-γ secretion was measured by ELISPOT analysis. Data shown represent means±s.d.'s of triplicates. Results are representative of three independent experiments. ns, not significant; * P <0.05 by unpaired, two-tailed Welch's t -test.

Article Snippet: Antibodies to the following proteins were used in immunoblots: mouse anti-Ii monoclonal antibodies (sc-6262, 1:1,000, and sc-47742, 1:500, Santa Cruz Biotechnology), mouse anti-DR/DP monoclonal antibody (sc-51617, 1:1,000, Santa Cruz Biotechnology), mouse anti-DR/DP monoclonal antibody (sc-51617, 1:2,000, Santa Cruz Biotechnology), mouse anti-DP monoclonal antibody (H127, 1:1,000, Leinco Technologies), mouse anti-DRα monoclonal antibody (sc-53499, 1:1,000, Santa Cruz Biotechnology), mouse anti-CLIP monoclonal antibody (sc-12725, 1:3,000, Santa Cruz Biotechnology), mouse anti-MAGE-A3 monoclonal antibody (H00004102-M0, 1:1,000, Novus Biologicals) and mouse anti-HLA class I monoclonal antibody (ab70328, 1:2,000, Abcam).

Techniques: Transfection, Cell Culture, Plasmid Preparation, Control, Expressing, Flow Cytometry, Enzyme-linked Immunospot, Two Tailed Test

Journal: Nature Communications

Article Title: HLA-DP 84Gly constitutively presents endogenous peptides generated by the class I antigen processing pathway

doi: 10.1038/ncomms15244

Figure Lengend Snippet: ( a , b ) The indicated K562-based aAPCs were transiently transfected with a retrovirus vector encoding IRES-EGFP (control) or a native ( a ) or endosome-targeted ( b ) form of MAGE-A3 linked with IRES-EGFP. Transient transfection efficiencies were normalized to EGFP expression measured by flow cytometry. DP4/MAGE-A3 243–258 CD4 + T cells were stimulated with the indicated APCs and IL-2 secretion was measured by ELISPOT analysis. Data shown represent means±s.d.'s of triplicates. ( c ) NSG mice were subcutaneously inoculated with 2 × 10 6 K562 cells stably expressing DP4/Ii/MAGE-A3 or DP4 84DEAV87 /Ii/MAGE-A3. Two days later, the mice were treated with 4 × 10 7 CD3 + T cells untransduced or transduced with DP4/MAGE-A3 243–258 TCR. The mean tumour size for each group is represented as the average±s.d. of three mice. There was no significant difference in the tumorigenicity of the two cell lines (data not shown). Results are representative of three independent experiments. ns, not significant; * P <0.05, ** P <0.01 by unpaired, two-tailed Welch's t -test.

Article Snippet: Antibodies to the following proteins were used in immunoblots: mouse anti-Ii monoclonal antibodies (sc-6262, 1:1,000, and sc-47742, 1:500, Santa Cruz Biotechnology), mouse anti-DR/DP monoclonal antibody (sc-51617, 1:1,000, Santa Cruz Biotechnology), mouse anti-DR/DP monoclonal antibody (sc-51617, 1:2,000, Santa Cruz Biotechnology), mouse anti-DP monoclonal antibody (H127, 1:1,000, Leinco Technologies), mouse anti-DRα monoclonal antibody (sc-53499, 1:1,000, Santa Cruz Biotechnology), mouse anti-CLIP monoclonal antibody (sc-12725, 1:3,000, Santa Cruz Biotechnology), mouse anti-MAGE-A3 monoclonal antibody (H00004102-M0, 1:1,000, Novus Biologicals) and mouse anti-HLA class I monoclonal antibody (ab70328, 1:2,000, Abcam).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Flow Cytometry, Enzyme-linked Immunospot, Stable Transfection, Transduction, Two Tailed Test

Journal: eLife

Article Title: Temporal transcription factors determine circuit membership by permanently altering motor neuron-to-muscle synaptic partnerships

doi: 10.7554/eLife.56898

Figure Lengend Snippet:

Article Snippet: Antibody , mouse anti-En (monoclonal) , 4D9 , DSHB Cat# 4D9 anti-engrailed/invected, RRID: AB_528224 , 1:5.

Techniques: Western Blot

Journal: The Journal of Neuroscience

Article Title: Slow Muscle Precursors Lay Down a Collagen XV Matrix Fingerprint to Guide Motor Axon Navigation

doi: 10.1523/JNEUROSCI.2847-15.2016

Figure Lengend Snippet: COLXV-B influences MP fate. A, Immunofluorescence of 24 hpf uninjected (uninj.) and MO15b-injected (MO15b) embryos with F59 (green) and collagen XII antibodies that stain vertical myosepta (COLXII, red). Orthogonal views at the level of the white line in A, B (right panels) show positions of migrated SSFs that were not affected in morphants (green). B, In situ hybridization of 18 hpf uninjected and MO15b-injected embryos with the MP marker engrailed2a (eng2a) probe. C, Immunostaining of 24 hpf uninjected and MO15b-injected embryos with the slow muscle marker Prox1 (red) and 4D9 (engrailed, green) antibodies that mark SSFs (red), MPs (yellow), and medial fast fibers (green) nuclei. D, Quantification of MP number (left) and total number of slow muscle cells (right, MPs + SSFs) per somite hemisegment of 24 hpf tMO15b-injected (n hemisegments = 56) and uninjected embryos (n hemisegments = 59) confirms an increase in the number of MPs in morphants. Data are mean ± SEM. NS, Nonsignificant. ***p < 0.001 (Student's t test). E, In situ hybridization of 18 hpf uninjected and MO15b-injected embryos with the patch2 receptor (ptch2) probe, a direct target of Hh signaling. Lateral views with anterior to the left. Cross sections were prepared at the position indicated by the dashed line in the corresponding lateral view.

Article Snippet: Primary and secondary antibodies and the drug α-bungarotoxin were used at indicated dilutions in 1% sheep serum in PBS-T: rabbit polyclonal anti-COLXV-B (1:100); rabbit polyclonal anti-COLXII (1:250) ( Bader et al., 2009 ); mouse monoclonal F59 antibody (1:10, Developmental Studies Hybridoma Bank [DSHB]); mouse monoclonal znp1 antibody (1:100, DSHB); mouse monoclonal zn8 antibody (1:400, DSHB); mouse monoclonal 4D9 antibody (1:50, DSHB); rabbit polyclonal Prox1 antibodies (1:5000, Millipore); α-bungarotoxin TRITC (10 μg/ml, Biolegend); goat anti-mouse or anti-rabbit IgG coupled to AlexaFluor-488 or AlexaFluor-546 (1:500, Invitrogen).

Techniques: Immunofluorescence, Injection, Staining, In Situ Hybridization, Marker, Immunostaining