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: Oncogene

Article Title: STAT5 represses BCL6 expression by binding to a regulatory region frequently mutated in lymphomas.

doi: 10.1038/sj.onc.1209775

Figure Lengend Snippet: Figure 1 STAT5 activation is associated with repression of BCL6. (a) BCL1 cells were treated with IL-2 for 15 min, and analysed by immunoblot for phosphorylated STAT5. Total STAT5 was used as a loading control. (b) BCL1 cells were untreated or treated with IL-2 for 2 h, and RNA was analysed by q real-time PCR (qPCR) for BCL6 and CIS expression normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (c) Starved NKL cells were untreated or stimulated with IL-2 for 15 min, and analysed by immunoblot for phosphorylated STAT5 and STAT5a. (d) Starved NKL cells were untreated or stimulated with IL-2 for 2 h, after which mRNA was analysed by qPCR for BCL6 and CIS expression normalized to b-actin as an invariant control. (e) Starved NKL cells were untreated or stimulated with IL-2 and analysed by immunoblot for BCL6 protein expression. Total STAT5 was used as a loading control. (f) Starved 32D cells were untreated or stimulated with IL-3 for 2 h, and RNA was analysed by qPCR as in (b). (g) K562 cells were untreated or treated with 1 mM STI-571 for 30 min. STAT5a and STAT5b were immunoprecipitated separately and analysed by immunoblot with the indicated antibodies. (h) K562 cells were untreated or treated with STI-571 for 2 h, and RNA was analysed by qPCR as in (d). (i) Starved Ba/f3-tet-1*6 cells were untreated or treated with dox, and RNA was analysed by qPCR as in (b).

Article Snippet: Antibodies used include phospho-specific STAT5 (9351), phospho-p44/42 MAPK (9101), p44/42 MAPK (9102), phospho-Akt (9271) and Akt (9272) from Cell Signaling; STAT5b (71-2500; Zymed, South San Francisco, CA, USA); and total STAT5 (sc-835), STAT5a (sc-1081), STAT1 (sc-346), STAT3 (sc-482) and BCL6 (sc-858) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). mRNA expression RNA was harvested using an RNeasy Mini Kit from Qiagen (Valencia, CA, USA). cDNA was generated using the SuperScript First Stand Synthesis kit (Invitrogen, Carlsbad, CA, USA) or the TaqMan Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA).

Techniques: Activation Assay, Western Blot, Control, Real-time Polymerase Chain Reaction, Expressing, Immunoprecipitation

Journal: Oncogene

Article Title: STAT5 represses BCL6 expression by binding to a regulatory region frequently mutated in lymphomas.

doi: 10.1038/sj.onc.1209775

Figure Lengend Snippet: Figure 2 (a) Inhibition of BCL6 by STAT5 relieves the repression on BCL6 target genes. Starved NKL cells were untreated or stimulated with IL-2 for 24 h, and RNA was analysed by qPCR normalized to GAPDH as an invariant control. (b) Inhibition of STAT5 activation results in BCL6 expression and repression of BCL6 target genes. K562 cells were untreated or treated with STI-571 for 24 h, and RNA was analysed by qPCR as in (a).

Article Snippet: Antibodies used include phospho-specific STAT5 (9351), phospho-p44/42 MAPK (9101), p44/42 MAPK (9102), phospho-Akt (9271) and Akt (9272) from Cell Signaling; STAT5b (71-2500; Zymed, South San Francisco, CA, USA); and total STAT5 (sc-835), STAT5a (sc-1081), STAT1 (sc-346), STAT3 (sc-482) and BCL6 (sc-858) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). mRNA expression RNA was harvested using an RNeasy Mini Kit from Qiagen (Valencia, CA, USA). cDNA was generated using the SuperScript First Stand Synthesis kit (Invitrogen, Carlsbad, CA, USA) or the TaqMan Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA).

Techniques: Inhibition, Control, Activation Assay, Expressing

Journal: Oncogene

Article Title: STAT5 represses BCL6 expression by binding to a regulatory region frequently mutated in lymphomas.

doi: 10.1038/sj.onc.1209775

Figure Lengend Snippet: Figure 3 Identification of potential STAT5-binding sites in the BCL6 gene. (a) A schematic representation of the BCL6 gene highlighting the different variant (V) untranslated exons and relative locations of region A and region B. The sequences of region A ( þ 8029 to þ 8130) (b) and region B ( þ 441 to þ 544) (c) of the BCL6 gene are shown with the STAT consensus sites underlined. Sequence numbers are in reference to the þ 1 nucleotide identified by Ohashi et al. (1995). (d) Quantitation of homology as percent identity of human BCL6 to mouse and rat BCL6 in the first exon and intron of BCL6 using the Multi-VISTA browser.

Article Snippet: Antibodies used include phospho-specific STAT5 (9351), phospho-p44/42 MAPK (9101), p44/42 MAPK (9102), phospho-Akt (9271) and Akt (9272) from Cell Signaling; STAT5b (71-2500; Zymed, South San Francisco, CA, USA); and total STAT5 (sc-835), STAT5a (sc-1081), STAT1 (sc-346), STAT3 (sc-482) and BCL6 (sc-858) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). mRNA expression RNA was harvested using an RNeasy Mini Kit from Qiagen (Valencia, CA, USA). cDNA was generated using the SuperScript First Stand Synthesis kit (Invitrogen, Carlsbad, CA, USA) or the TaqMan Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA).

Techniques: Binding Assay, Variant Assay, Sequencing, Quantitation Assay

Journal: Oncogene

Article Title: STAT5 represses BCL6 expression by binding to a regulatory region frequently mutated in lymphomas.

doi: 10.1038/sj.onc.1209775

Figure Lengend Snippet: Figure 4 Region B of the BCL6 gene is STAT5 responsive. 293 cells were transfected with A-Luc or B-Luc (a) or TBR-Luc (b), and STAT5a1*6 or vector control, and analysed for luciferase activity after 24 h.

Article Snippet: Antibodies used include phospho-specific STAT5 (9351), phospho-p44/42 MAPK (9101), p44/42 MAPK (9102), phospho-Akt (9271) and Akt (9272) from Cell Signaling; STAT5b (71-2500; Zymed, South San Francisco, CA, USA); and total STAT5 (sc-835), STAT5a (sc-1081), STAT1 (sc-346), STAT3 (sc-482) and BCL6 (sc-858) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). mRNA expression RNA was harvested using an RNeasy Mini Kit from Qiagen (Valencia, CA, USA). cDNA was generated using the SuperScript First Stand Synthesis kit (Invitrogen, Carlsbad, CA, USA) or the TaqMan Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay

Journal: Blood Advances

Article Title: Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma

doi: 10.1182/bloodadvances.2021005682

Figure Lengend Snippet: FBXO11 tumor-derived mutants show impaired ability to induce BCL6 and SNAIL degradation. (A) Representative western blot for FBXO11 and BCL6 protein expression in HEK-293T cells transfected with BCL6 in combination with GFP, WT FBXO11, or FBXO11 mutants at the indicated time points after treatment with CHX. FBXO11 mutants were detected by anti-FBXO11 antibody, and BCL6 was detected by anti-BCL6 antibody. β-actin was used as a loading control. GFP was used for transfection efficiency. One representative experiment of 2 independently performed experiments is shown. (B) Impaired degradation of SNAIL by FBXO11 mutants. Representative western blot for FBXO11 and SNAIL protein expression in HEK-293T cells transfected with SNAIL and WT FBXO11 or the indicated FBXO11 mutants at the indicated time points after treatment with CHX. FBXO11 mutants were detected by anti-Flag antibody. β-actin was used as a loading control. One representative experiment of 2 independently performed experiments is shown. (C) Degradation kinetics of BCL6 by FBXO11 mutants. BCL6 abundance was measured from western blot gels as in (A) by ImageJ software and normalized for the GFP intensity of the corresponding lane. The ratio between the relative levels of BCL6/GFP at each time 0 was set as 100%. Data are from 1 of 2 independent experiments, each with comparable results. (D) SNAIL abundance was measured from western blot gels, as in (B), using ImageJ software and normalized for β-actin intensity of the corresponding lane. The ratio between the relative levels of SNAIL/β-actin at each time 0 was set as 100%. Data are from 1 of 2 independent experiments, each with comparable results.

Article Snippet: FLAG-tagged SNAIL (clone ID: 16218) and FLAG-tagged BCL6 (clone ID: 31391) plasmids were purchased from Addgene (Cambridge, MA).

Techniques: Derivative Assay, Western Blot, Expressing, Transfection, Software

Journal: Blood Advances

Article Title: Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma

doi: 10.1182/bloodadvances.2021005682

Figure Lengend Snippet: FBXO11 deletion accelerates lymphomagenesis in Eμ-myc–transgenic mice. (A) Kaplan-Meier survival analysis of Eμ-myc (n = 33), Eμ-myc/FBXO11fl/fl;CD19-Cretg/+ (KO) (n = 21) and Eμ-myc/FBXO11fl/+;CD19-Cretg/+ (HET) (n = 16) mice, with a median survival time of 121, 89, and 85 days, respectively. Eμ-myc (n = 33) are a combination of Eμ-myc/FBXO11fl/fl (n = 23) and Eμ-myc/FBXO11fl/+ (n = 10). P < .0001 log-rank (Mantel-Cox) test. (B) B220 and IgM expression in lymphomas from Eμ-myc (n = 17), Eμ-myc/FBXO11fl/fl;CD19-Cretg/+ (KO) (n = 16), and Eμ-myc/FBXO11fl/+;CD19-Cretg/+ (HET) (n = 9) mice. (C) B220 and IgD expression in lymphomas from Eμ-myc (n = 16), Eμ-myc/FBXO11fl/fl;CD19-Cretg/+ (KO) (n = 10), and Eμ-myc/FBXO11fl/+;CD19-Cretg/+ (HET) (n = 5) mice. (D) Representative flow cytometry of B220 and IgM expression in lymphoma from Eμ-myc, Eμ-myc/FBXO11-KO, and Eμ-myc/FBXO11 HET mice. (E) Representative H&E stains (top row) and immunohistochemistry for cleaved caspase 3 (middle row) and Ki-67 (bottom row) performed on Eμ-myc primary lymphoma with the indicated genotypes (original magnification ×200). Scale bars, 50 μm. Insets show high-magnification images. (F) Quantification of macrophages in Eμ-myc lymphoma with the indicated genotypes (n = 6 mice for each genotype). Data are mean (± standard deviation [SD]) macrophages per 40× field. Quantification of cleaved caspase 3+ (G) and Ki-67+ (H) cells in Eμ-myc lymphoma with the indicated genotypes (n = 4 mice for each genotype). Data are mean ± SD. (I) BCL6 stability in Eμ-myc/FBXO11-KO lymphoma cells. Western blot showing BCL6 protein expression after treatment with CHX for the indicated times in 3 immortalized Eμ-myc/FBXO11fl/fl lymphoma cell lines transduced with a tamoxifen-inducible CreERT2 vector. Cre recombinase was activated by treatment with 10 nM 4-hydroxytamoxifen (4-OHT) for 4 hours to induce deletion of the FBXO11 floxed gene. A20 is a mouse BCL6+ B lymphoma cell line used as control. **P < .01, ***P < .001, unpaired 2-tailed Student t test.

Article Snippet: FLAG-tagged SNAIL (clone ID: 16218) and FLAG-tagged BCL6 (clone ID: 31391) plasmids were purchased from Addgene (Cambridge, MA).

Techniques: Transgenic Assay, Expressing, Flow Cytometry, Immunohistochemistry, Standard Deviation, Western Blot, Transduction, Plasmid Preparation

Journal: Blood Advances

Article Title: Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma

doi: 10.1182/bloodadvances.2021005682

Figure Lengend Snippet: FBXO11 deletion in BL cell lines leads to BCL6 protein stabilization. (A) BLC6 stability in human BL lines in which FBXO11 is knocked out. Representative western blot showing BCL6 protein expression in 3 independent FBXO11 KO RAJI cell lines (clone #1 obtained with sgRNA#1, clones #2 and #3 obtained with sgRNA#2; supplemental Figure 5B-E) after treatment with CHX for the indicated times. FARAGE cell line, which is homozygous for deletion of the FBXO11 gene, was used as control for BCL6 stability. One representative experiment of 3 independently performed experiments is shown. (B) Representative western blot showing BCL6 protein expression in WT or FBXO11-KO DAUDI cell line after treatment with CHX for the indicated times. β-actin was used as a loading control. One representative experiment of 3 independently performed experiments is shown. (C) Degradation kinetics of BCL6 in BL WT or FBXO11-KO cells. BCL6 abundance was measured from western blot gels as in (A-B) using ImageJ software and normalized for the β-actin intensity of the corresponding lane. (D) Quantitative real-time PCR expression analysis of the BCL6 target genes DUSP5, CDKN1A, and CD69 mRNA in RAJI and 2 FBXO11-KO RAJI cell lines (#1 and #2). Data are mean ± standard deviation. (E) Hierarchical clustering (Euclidean distance, average linkage) of 172 genes detected as differentially expressed between the FBXO11-WT or -KO RAJI cell lines. (F) Analysis of genes detected as differentially expressed between WT cell lines and KO clones. Forty-seven of 172 differentially expressed genes can be connected to each other using Ingenuity Pathway Analysis (QIAGEN IPA) in a BCL6-centered network. **P < .01, ***P < .001, unpaired 2-tailed Student t test.

Article Snippet: FLAG-tagged SNAIL (clone ID: 16218) and FLAG-tagged BCL6 (clone ID: 31391) plasmids were purchased from Addgene (Cambridge, MA).

Techniques: Western Blot, Expressing, Clone Assay, Software, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Blood Advances

Article Title: Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma

doi: 10.1182/bloodadvances.2021005682

Figure Lengend Snippet: BCL6 degradation impairs BL growth and potentiates MYC inhibition. (A) Western blot analyses for BCL6 expression on the indicated BL cell lines. Cells were treated for 60 minutes with BI-3802 (1 or 5 μM), BI-3812 (1 μM), or vehicle (-) before collection. Data are representative of 2 experiments. β-actin was used as loading control. (B) Western blot analysis for BCL6 on immortalized lymphoma cell lines obtained from FBXO11-WT (Eμ-myc) or -KO (Eμ-myc/FBXO11fl/fl;CD19-Cretg/+) mice. Cells were treated for 1 hour with BI-3802 (1 or 5 μM), BI-3812 (1 μM), or vehicle (-) before collection. Data are representative of 2 experiments with similar results. β-actin was used as loading control. (C) Effects of BCL6 degradation on WT and FBXO11-KO RAJI (clones #1 and #2) cell growth. Cells were kept at constant concentrations of the degrader BI-3802 as indicated (1 μM or 5 μM) and split to 100 000 cells per milliliter every 3 days. Split rates were multiplied to derive growth curves. Data are mean ± standard error of the mean (SEM) and are representative of ≥3 independent experiments. *P < .05, **P < .01, unpaired 2-tailed Student t test. (D) Growth of tumor xenografts in NSG mice of WT and FBXO11-KO RAJI (clones #1 and #2) cells treated with BI-3802 (5 μM) for 24 hours before injection. Data (n = 5 mice per group) are shown as mean ± SEM. **P < .01, paired 2-tailed Student t test. (E) Growth curves of WT and FBXO11-KO RAJI (clones #1 and #2) cells treated with Omomyc mini-protein (2 or 5 μM), alone or in combination with BI-3802 (5 μM). Total cell number was quantified at the indicated time points. Data are mean ± SEM of triplicates from a single experiment representative of 2 repeats. **P < .01, ***P < .001, unpaired 2-tailed Student t test. (F) Growth curves of WT and FBXO11-KO RAJI (clones #1 and #2) cells infected with a doxycycline-inducible lentiviral vector expressing Omomyc. Cells were treated only with doxycycline to induce Omomyc expression (Omomyc), or BI-3802 (5 μM) or a combination of doxycycline and BI-3802 (Omomyc + BI-3802). Total cell number was quantified at the indicated time points. Data are mean ± SEM of triplicates from a single experiment representative of two repeats. **P < .01, ***P < .001, unpaired 2-tailed Student t test.

Article Snippet: FLAG-tagged SNAIL (clone ID: 16218) and FLAG-tagged BCL6 (clone ID: 31391) plasmids were purchased from Addgene (Cambridge, MA).

Techniques: Inhibition, Western Blot, Expressing, Clone Assay, Injection, Infection, Plasmid Preparation

Journal: Blood Advances

Article Title: Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma

doi: 10.1182/bloodadvances.2021005682

Figure Lengend Snippet: Combined targeting of MYC and BCL6 severely impairs BL growth in mice by reducing proliferation and increasing apoptosis. (A) Growth of tumor xenografts of RAJI cells transduced with doxycycline-inducible Omomyc vector in NSG mice. Lymphoma cells were treated with BI-3802 (5 μM) for 24 hours before injection. Mice were administered normal water (red line) or doxycycline water (blue line) from day 0. Data are mean ± standard error of the mean of 8 tumors per group. *P < .05, paired 2-tailed Student t test. (B) Representative H&E stains (top row) and immunohistochemistry for cleaved caspase 3 (middle row) and Ki-67 (bottom row) performed on RAJI lymphoma arising in NSG mice and treated with the BCL6 degrader BI-3802 together with Omomyc, as in (A) (original magnification ×200). Scale bars, 50 μm. Quantification of cleaved caspase 3+ (C) and Ki-67+ (D) cells in RAJI lymphoma with the indicated treatment. Data were obtained from 4 areas in 4 independent tumors for each treatment. Data are mean ± standard deviation. **P < .01, unpaired 2-tailed Student t test. (E) Representative immunofluorescence performed on RAJI lymphoma grafted in NSG mice and treated with the BCL6 degrader BI-3802 together with Omomyc, as in (A). Cells were stained for Ki-67 (green), Omomyc (red), and DAPI (blue) for the nucleus (original magnification, ×200). Scale bars, 100 μm. Higher-magnification images of the white boxes are shown in the far right panels. Pink arrow-heads in inset images indicate Ki-67−/Omomyc+ nuclei. (F) Quantification of the experiment described in (E). Three sections for each treatment were used to score the number of Ki-67+/Omomyc− and Ki-67+/Omomyc+ nuclei. ***P < .001.

Article Snippet: FLAG-tagged SNAIL (clone ID: 16218) and FLAG-tagged BCL6 (clone ID: 31391) plasmids were purchased from Addgene (Cambridge, MA).

Techniques: Transduction, Plasmid Preparation, Injection, Immunohistochemistry, Standard Deviation, Immunofluorescence, Staining