Journal: The Journal of Immunology Author Choice

Article Title: Defining T Cell Subsets in Human Tonsils Using ChipCytometry

doi: 10.4049/jimmunol.2100063

Figure Lengend Snippet: Intracellular markers for ChipCytometry cell suspension staining

Article Snippet: 1 , BCL6 , REA373 , PE , Miltenyi Biotec.

Techniques: Suspension

Journal: The Journal of Immunology Author Choice

Article Title: Defining T Cell Subsets in Human Tonsils Using ChipCytometry

doi: 10.4049/jimmunol.2100063

Figure Lengend Snippet: Phenotyping of tonsil-derived, CD3-enriched cells by cell suspension ChipCytometry. Four human tonsils were processed to single-cell suspensions, positively enriched for CD3 + T cells, barcoded, loaded onto a microfluidic chip, fixed, and subsequently analyzed by ChipCytometry with a panel of 19 fluorochrome-labeled Abs. ( A ) Density plot shows t -SNE analysis of concatenated samples, including all CD3 + CD19 − cells. ( B ) Clusters that were identified via FlowSOM were projected onto the t -SNE coordinates and color coded as indicated. ( C ) A heatmap showing median marker intensities within each cell cluster was generated to characterize cell clusters and derive cell labels. The heatmap color represents median marker expression across all four aggregated samples. ( D ) T-SNE plots show the expression of 16 markers within the aggregated samples. ( E ) Top panel, Within CD4 + T cells, the subsets TFH, pre-TFH, and non-TFH cells were identified according to expression of CXCR5 and PD1. Center panel, BCL6 expression is shown within non-TFH (red), pre-TFH (blue), and TFH (orange) cell subsets in human tonsils. Bottom panel, T-SNE plot shows non-TFH (red), pre-TFH (blue), TFH (orange), and non-CD4 T cells (black). Plots in (E) are derived from one donor and are representative of four donors.

Article Snippet: 1 , BCL6 , REA373 , PE , Miltenyi Biotec.

Techniques: Derivative Assay, Suspension, Labeling, Marker, Generated, Expressing

Journal: Endocrine Oncology

Article Title: Vitamin D receptor expression in germinal centre type diffuse large B-cell lymphoma cells is associated with vitamin D insensitivity

doi: 10.1530/EO-25-0057

Figure Lengend Snippet: Vitamin D receptor (VDR) protein is expressed in both GC- and ABC-type DLBCL cells. (A and B) VDR transcript expression (probe 840) and BCL6 transcript expression in cultured human DLBCL cell lines, determined by 2 −ΔΔCt real-time PCR analysis after normalisation to 18S , relative to expression in DB, n = 3, mean ± SD. (C and D) Western blot analysis of human DLBCL cell lines. GC- and ABC-DLBCL blots in C were developed in parallel, representative of at least two experiments. (E) Double immunohistochemistry detecting VDR (brown) and CD20 (blue) staining of OCI-Ly8 cells. (F) DepMap analysis of VDR and BCL6 transcript expression in 30 DLBCL cell lines subtyped as GC (black), ABC (grey) or unknown (white).

Article Snippet: Universal MasterMix II (LifeTechnologies) and the following TaqMan assays (all Applied Biosystems, UK) were used for quantitative amplification on a Chromo4 instrument (BioRad, UK): hVDR (Hs01045840_m1), hIRF4 (Hs00180031_m1), hIGF1 (Hs01547656_m1), hHPGD (Hs00960587_m1), hSLA (Hs00277129_m1), hBCL6 (Hs00153368_m1) and hIL6 (Hs00985639_m1).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 2. Experimental design. The BCL6 protein complex was iso- lated by antibody immunoprecipitation, and the proteins were sepa- rated by SDS-PAGE. The entire lane was submitted, in 12 separate gel slices, for trypsin digestion and analysis by ion trap tandem mass spectrometry.

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques: Immunoprecipitation, SDS Page, Mass Spectrometry

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 3. Immunoprecipitation of BCL6. Proteins immunoprecipi- tated from total cell lysates of SUDHL-4 and SUDHL-9 cell lines using a polyclonal anti-BCL6 antibody or rabbit IgG control were divided and resolved on parallel 8% SDS-polyacrylamide gels. A, silver stain- ing of one gel demonstrated a 95-kDa band corresponding to BCL6 that was present in the anti-BCL6 immunoprecipitate of SUDHL-4 lysates but not in the IgG immunoprecipitation control or the anti- BCL6 immunoprecipitate of SUDHL-9 lysates. B, after electrophore- sis, the proteins in the second gel were transferred to nitrocellulose membrane and immunoblotted using anti-BCL6 monoclonal anti- body. A band at 95 kDa corresponding to BCL6 was identified in the SUDHL-4 total cell lysate but not the SUDHL-9 or IgG control lanes.

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques: Immunoprecipitation, Control, Silver Staining, Membrane

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 1. BCL6-interacting proteins. The diagram shows the con- served motifs that mediate BCL6 protein interactions with the pro- teins listed under each domain. BCL6 directly interacts with several proteins via the POZ/BTB domain, the zinc finger domain, or both. p300 binds and acetylates BCL6, targeting it for degradation.

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques:

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 4. Protein identification by tan- dem mass spectrometry. MS/MS se- quencing of peptides identifying the pro- teins BCL6 (A), BCL11 (B), and PKC- (C). The MS full scan is shown for each sample followed by data-dependent MS/MS sequencing scans of a top pep- tide candidate.

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques: Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 5. Western blot and reciprocal immunoprecipitation valida- tion. A, proteins immunoprecipitated from SUDHL-4 lysates using anti-BCL6 were subjected to Western blotting using antibodies to the previously reported BCL6-interacting proteins HDAC4 and N-CoR2/ SMRT and the novel interacting proteins PKC- and WNK4. The bands were similar to those present in total cell lysates but were not seen in IgG immunoprecipitation controls. B, proteins immunoprecipi- tated from SUDHL-4 lysates using polyclonal antibodies to HDAC4 (previously reported BCL6-interacting protein) and PKC (novel inter- acting protein) were subjected to Western blotting using anti-BCL6. Bands correlating to BCL6 were identified by Western blotting in both pull-downs but not in the IgG control. IP, immunoprecipitation; WB, Western blot.

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques: Western Blot, Immunoprecipitation, Control

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 6. Cellular location and molecular function of BCL6-inter- acting proteins. The relative proportions of identified proteins are shown for the subcellular localization (A) and functional classification (B).

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques: Functional Assay

Journal: Theranostics

Article Title: Tumor suppressive BTB/POZ zinc-finger protein ZBTB28 inhibits oncogenic BCL6/ZBTB27 signaling to maintain p53 transcription in multiple carcinogenesis

doi: 10.7150/thno.34983

Figure Lengend Snippet: ZBTB28 and BCL6 inhibit and depend on each other. (A) ZBTB28 and BCL6 expression in multiple carcinoma cells. (B) Inverse association of ZBTB28 and BCL6 expression in lung cancer and ESCC, obtained from TCGA cancer dataset, accessed through cBioPortal ( www.cbioportal.org ). (C) Co-localization of BCL6 and ZBTB28 as determined using confocal microscopy. (D) Co-IP outcome revealing the binding between BCL6 and ZBTB28. Anti-HA (#ab1424, Abcam), BCL6 (sc-7388; Santa Cruz, Germany) or IgG (#2729, Cell Signaling Technology) antibodies were used. (E) ZBTB28 inhibits BCL6 promoter activities, as detected by dual luciferase reporter system. (F) qRT-PCR results of BCL6 mRNA expression in HONE1, KYSE150, and A549 cells. Cells were transfected with vector and ZBTB28 with or without BCL6. (G) qRT-PCR of BCL6 mRNA in A549 underwent with vector, BCL6 siRNA, and ZBTB28 with or without BCL6 siRNA. (H) Knock-down BCL6 by siRNA increased ZBTB28 mRNA expression levels. (I) Structure of wild type and mutant ZBTB28 reporter plasmid. (J) Promoter luciferase activity of wild-type and mutant ZBTB28 in HONE1 cells. Cells were transfected with BCL6, or ZBTB28 or both plasmids. (K) BCL6 inhibits ZBTB28 reporter activity in BCL6-null K562 cells. (L) The interdependent role of ZBTB28 and BCL6 was detected using MTS assay in a tumor cell line which lack of ZBTB28 expression.

Article Snippet: The diluted samples were incubated with HA antibody (#3274, Cell Signaling Technology), or BCL6 antibody (#14895, Cell Signaling Technology) overnight at 4°C, followed by capture with protein A/G magnetic beads (#9006, Cell Signaling Technology) for 2 h. Histone H3 antibody (#4620, Cell Signaling Technology) and Normal rabbit IgG (#2729, Cell Signaling Technology) were used as positive and negative controls, respectively.

Techniques: Expressing, Confocal Microscopy, Co-Immunoprecipitation Assay, Binding Assay, Luciferase, Quantitative RT-PCR, Transfection, Plasmid Preparation, Knockdown, Mutagenesis, Activity Assay, MTS Assay

Journal: Theranostics

Article Title: Tumor suppressive BTB/POZ zinc-finger protein ZBTB28 inhibits oncogenic BCL6/ZBTB27 signaling to maintain p53 transcription in multiple carcinogenesis

doi: 10.7150/thno.34983

Figure Lengend Snippet: ZBTB28 directly promotes p53 transcription in p53-wt cell lines. (A) Data from the TCGA online database confirmed a positive relationship between ZBTB28 and p53 expression in tumor cells. (B) The expression of p53 and its downstream target genes as detected by qRT-PCR in ZBTB28-overexpressing and control cells. (C) The expression of p53 and its downstream target genes as detected by qRT-PCR in A549 cells with ZBTB28 overexpression (* p <0.05, ** p <0.01, *** p <0.001, vector vs ZBTB28). (D) Locations of ChIP PCR primers (for segment 1, 2 and 3) at the p53 promoter and structure of the p53 promoter-luciferase reporter construct. The bottom panel is the consensus binding sequence motif of ZBTB28. (E) ChIP analysis with HA-tag or BCL6 antibody, for ZBTB28- or BLC6 binding to the p53 promoter, in HONE1 cells. ChIP with a mouse IgG antibody was used as a negative control. ZBTB28 competes for BLC6 for its binding to the p53 promoter. (F) ZBTB28 activates the p53 promoter activity, detected using a dual luciferase reporter system.

Article Snippet: The diluted samples were incubated with HA antibody (#3274, Cell Signaling Technology), or BCL6 antibody (#14895, Cell Signaling Technology) overnight at 4°C, followed by capture with protein A/G magnetic beads (#9006, Cell Signaling Technology) for 2 h. Histone H3 antibody (#4620, Cell Signaling Technology) and Normal rabbit IgG (#2729, Cell Signaling Technology) were used as positive and negative controls, respectively.

Techniques: Expressing, Quantitative RT-PCR, Control, Over Expression, Plasmid Preparation, Luciferase, Construct, Binding Assay, Sequencing, Negative Control, Activity Assay