bcl6 ko  (Santa Cruz Biotechnology)

Journal: European journal of immunology

Article Title: Inhibition of stearoyl-CoA desaturases suppresses follicular help T- and germinal center B- cell responses

doi: 10.1002/eji.201948257

Figure Lengend Snippet: Human and mouse TFH express higher levels of SCD mRNA. (A) Relative SCD1 expression in published microarray data (GEO# GSE50391) CD45RO+CXCR5− (non-TFH), CD45RO+CXCR5int (TFH), and CD45RO+CXCR5hi (GC-TFH) cells from human tonsils (five donors per group). (B) Relative expression of Fasn, Scd1, and Scd2 in published microarray data (GEO# GSE40068) from BCL6hiCXCR5+ (TFH) and BCL6−CXCR5− (non-TFH) cells of the mice immunized with KLH in CFA (two samples each pooled from three mice per group). (C) Relative expression of Fasn, Scd1, Scd2, and Bcl6 in published RNAseq data (GEO# GSE124883) from LN, spleen, or blood TFH and conventional T cells (Tconv) following NP-OVA immunization (three samples each pooled form ten mice per group). (D) Relative expression of Fasn, Scd1, and Scd2 in sorted splenic TFH (PD-1+CXCR5+) and non-TFH (PD1−CXCR5−) cells isolated from X31 immunized mice at 14 d.p.i. Representative results of (D) obtained from two independent experiments (five mice per groups). Results are given as mean ± SEM with one- (A) or two- (C) way ANOVA or unpaired t-test (D). *indicates significant differences (p < 0.05), **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Western blot analysis Naïve CD4 + T cells were purified from WT or BCL6 KO mice with naïve CD4 + T-cell isolation kit (Miltenyi Biotec).

Techniques: Expressing, Microarray, Isolation

Journal: European journal of immunology

Article Title: Inhibition of stearoyl-CoA desaturases suppresses follicular help T- and germinal center B- cell responses

doi: 10.1002/eji.201948257

Figure Lengend Snippet: BCL6 promotes SCD1 expression. (A) Relative expression of Scd1 and Scd2 in WT or BCL6 KO CD4+ T cells under the TFH cell polarizing condition for 4 days. (B) Western blot analysis of SCD1 protein levels in WT or BCL6 KO CD4+ T cells under the TFH cell polarizing condition for 5 days. (C) Scd1, Scd2, and Bcl6 expression in the BCL6-retroviral transduced CD4+ T cells under the TFH cell polarizing condition. (D) WT or BCL6 KO-OTII cells were transfer into CD45.1 congenic mice at one day before X31-OTII virus immunization. After day 14 postimmunization, the OTII cells were sorted and then Scd1 and Scd2 were measured by quantitative RT-PCR (three mice per group in two independent experiments). Representative graphs are obtained from at least two (A, B, and C) independent experiments (two mice per group). Results are given as mean ± SEM with unpaired t-test: *p < 0.05; **p < 0.01; and ***p < 0.001.

Article Snippet: Western blot analysis Naïve CD4 + T cells were purified from WT or BCL6 KO mice with naïve CD4 + T-cell isolation kit (Miltenyi Biotec).

Techniques: Expressing, Western Blot, Retroviral, Virus, Quantitative RT-PCR

Journal: Journal of the American Chemical Society

Article Title: Rewiring the fusion oncoprotein EWSR1::FLI1 in Ewing sarcoma with bivalent small molecules

doi: 10.1021/jacs.5c05634

Figure Lengend Snippet: Volcano plots of differentially expressed RNA species in BCL6 KO (two guides averaged over three replicates per guide) EWS502 cells (A) or TC32 cells (B) vs Chr2.2 control cells. GSEA in EWS502 (C) or TC32 (D) BCL6 KO cells shows a positive correlation with a published BCL6 gene signature derived from BCL6 promoter binding data 25 . SOCS2 and CISH transcripts have increased expression by RT-qPCR in BCL6 KO EWS502 cells (E and F) or TC32 cells (G and H) vs control guides. Average expression from three independent cell transductions (performed in technical triplicate) is shown. RT-qPCR data was compared by one-way ANOVA; NS = not significant, **** p < 0.001. All error bars in the figure show mean ± SD. Immunoblotting shows BCL6 KO and corresponding increase in SOCS2 protein levels in EWS502 (I) and TC32 (J) cells. Each lane is from an independent transduction of cells. GAPDH serves as a loading control.

Article Snippet: For BCL6 KO experiments, 2 × 10 6 EWS502 or TC32 cells were seeded into 6-well plates in a volume of 1 mL of RPMI media supplemented with 8 or 4 μg/mL of polybrene (Santa Cruz Biotechnology, SC-134220), respectively.

Techniques: Control, Derivative Assay, Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Transduction

Journal: Journal of the American Chemical Society

Article Title: Rewiring the fusion oncoprotein EWSR1::FLI1 in Ewing sarcoma with bivalent small molecules

doi: 10.1021/jacs.5c05634

Figure Lengend Snippet: (A) Schematic of EB-TCIP mechanism of action. EB-TCIP induces a ternary complex between FKBP F36V tagged EWSR1::FLI1 and BCL6, which leads to activation of BCL6 target gene transcription. Image made with Biorender. (B) Structures of compounds used in this work. (C) EB-TCIP increases the association of BCL6 with FKBP-E::F in a dose-dependent manner in EWS502 FKBP-E::F cell lysates while NEG-1 (D) does not induce a ternary complex. The association is reversible as excess BI3812 (C) and excess OAP (free acid) (D) abrogate ternary complex formation. GAPDH was probed to confirm that unbound proteins were removed by washing. EB-TCIP dose-dependently increases SOCS2 (E) and CISH (F) expression by RT-qPCR. (G) SOCS2 protein levels dose-dependently increase while BCL6 protein levels dose-dependently decrease after EB-TCIP treatment. EB-TCIP induces higher SOCS2 (H) and CISH (I) transcript levels than chemical inhibition with BI3812 or chemically induced degradation with BI3802 (DEG) . (J) SOCS2 protein levels are highest in EB-TCIP treated cells compared to BI3812 , BI3802 (DEG) , or negative control compounds that do not form ternary complexes. Immunoblotting is representative of three biological replicates and GAPDH serves as a loading control. All experiments were performed in FKBP-E::F expressing EWS502 cells. RT-qPCR experiments show one experiment with technical triplicate that is representative of three biological replicates. Means of SOCS2 and CISH expression were compared using one-way ANOVA with multiple comparisons; NS = not significant, *** p < 0.005, **** p < 0.001. All error bars in the figure indicate mean ± SD. Unless indicated with brackets, significance above each condition indicates comparison of that mean to the mean of DMSO.

Article Snippet: For BCL6 KO experiments, 2 × 10 6 EWS502 or TC32 cells were seeded into 6-well plates in a volume of 1 mL of RPMI media supplemented with 8 or 4 μg/mL of polybrene (Santa Cruz Biotechnology, SC-134220), respectively.

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Inhibition, Negative Control, Western Blot, Control, Comparison

Journal: Journal of the American Chemical Society

Article Title: Rewiring the fusion oncoprotein EWSR1::FLI1 in Ewing sarcoma with bivalent small molecules

doi: 10.1021/jacs.5c05634

Figure Lengend Snippet: (A) Time course of SOCS2 and BCL6 protein levels. BCL6 degradation occurs within 1 h for both EB-TCIP and BI3802 (DEG) . EB-TCIP induces SOCS2 expression by 2 h and maintains higher expression levels than BI3812 or BI3802 (DEG) throughout the time course. SOCS2 (B) and CISH (C) transcripts reach a maximum between 2 and 4 h by RT-qPCR. (D) EB-TCIP induced SOCS2 protein expression can be reversed with 25-fold excess OAP (free acid). Co-treatment of 1 µM BI3812 and OAP do not increase SOCS2 protein expression more than 1 µM BI3812 alone. EB-TCIP induced SOCS2 (E) and CISH (F) transcript expression is reversed with excess OAP . BI3812 and OAP must be chemically linked to induce maximum transcript expression. (G) EB-TCIP induces the highest expression of SOCS2 protein in EWS502 FKBP-E::F cells compared to EWS502 parental cells or EWS502 cells expressing FKBP-GFP. Only treatment with EB-TCIP induces more expression of SOCS2 (H) and CISH (I) than BI3812 in EWS502 FKBP-E::F cells. EB-TCIP : BI3812 ratio was calculated by dividing the average expression of each transcript in EB-TCIP treated cells by the average expression of each transcript in BI3812 treated cells. Immunoblotting is representative of three biological replicates. RT-qPCR experiments show one experiment with technical triplicate or quadruplicate that is representative of three biological replicates. The means of SOCS2 and CISH expression were compared using one-way ANOVA with multiple comparisons; NS = not significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001. All error bars in the figure represent mean ± SD. Unless indicated with brackets, significance above each condition indicates comparison of that mean to the mean of DMSO. In (H) and (I), unless indicated with brackets, the means of BI3812 , EB-TCIP , and NEG-1 were compared to the DMSO sample for the corresponding cell line.

Article Snippet: For BCL6 KO experiments, 2 × 10 6 EWS502 or TC32 cells were seeded into 6-well plates in a volume of 1 mL of RPMI media supplemented with 8 or 4 μg/mL of polybrene (Santa Cruz Biotechnology, SC-134220), respectively.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Comparison

Journal: Journal of the American Chemical Society

Article Title: Rewiring the fusion oncoprotein EWSR1::FLI1 in Ewing sarcoma with bivalent small molecules

doi: 10.1021/jacs.5c05634

Figure Lengend Snippet: Volcano plots portraying log 2 fold changes of gene expression from cells treated with 2.5 µM EB-TCIP versus DMSO at 8 (A) and 24 (B) h with a −log 10 adjusted P-value cut off of 1. EB-TCIP treatment predominantly increases expression of transcripts at both timepoints. Volcano plots portraying log 2 fold changes of cells treated with 2.5 µM EB-TCIP versus 2.5 µM BI3812 (C) or 2.5 µM NEG-1 (D) at 8 hours with a −log 10 P-value cut off of 1.3. EB-TCIP induces higher expression of BCL6 transcripts than BI3812 or NEG-1 at this early timepoint. Dots corresponding to SOCS2, CISH, and CXCL11 are labelled with black borders. (E) Heatmaps of changes in BCL6 target gene expression at 4, 8 and 24 h show that EB-TCIP induces faster and/or higher expression of these select genes. (F) GSEA comparing EB-TCIP treated EWS502 FKBP-E::F cells to BCL6 KO EWS502 parental cells (LFC ≥ 1.65) at 4 (top), 8 (middle) and 24 h (bottom) show significant positive correlation between the two gene sets. RNA-seq data is shown as the average of three independent replicates.

Article Snippet: For BCL6 KO experiments, 2 × 10 6 EWS502 or TC32 cells were seeded into 6-well plates in a volume of 1 mL of RPMI media supplemented with 8 or 4 μg/mL of polybrene (Santa Cruz Biotechnology, SC-134220), respectively.

Techniques: Gene Expression, Expressing, Targeted Gene Expression, RNA Sequencing

Journal: Journal of the American Chemical Society

Article Title: Rewiring the fusion oncoprotein EWSR1::FLI1 in Ewing sarcoma with bivalent small molecules

doi: 10.1021/jacs.5c05634

Figure Lengend Snippet: (A) ChIP-seq tornado plots of HA (FKBP-E::F) binding signal of EB-TCIP (red) versus DMSO (black) peaks that are decreasing (DEC; 92), non-significantly changing (NS; 8656), and increasing (INC; 2296) at 24 h. Differential peaks between EB-TCIP and DMSO are shown for all compounds. Compared to BI3812 (brown) and BI3802 ( DEG , purple), EB-TCIP increases FKBP-E::F binding at a subset of genes. Line plots for all compound treatments in each cluster are shown to the right. (B) Scatter plot portraying top enriched motifs of HA binding sites in DMSO treated cells. (C) Scatter plot portraying top motifs of HA increased peaks enriched in EB-TCIP treated cells. The BCL6 motif scores 29 th . (D) Scatter plot portraying top motifs of HA decreased peaks enriched in EB-TCIP treated cells. IGV visualization of input, HA (FKBP-E::F), BCL6, ATAC-seq signal, and RNA-seq signal at the SOCS2 (E) and CISH (F) with treatments DMSO (black), 1 µM BI3812 (brown), 1 µM BI3802 (DEG , purple), and 1 µM EB-TCIP (red). All ChIP-seq and ATAC-seq is portrayed as the average of two independent replicates. RNA-seq is portrayed as the average of three independent replicates.

Article Snippet: For BCL6 KO experiments, 2 × 10 6 EWS502 or TC32 cells were seeded into 6-well plates in a volume of 1 mL of RPMI media supplemented with 8 or 4 μg/mL of polybrene (Santa Cruz Biotechnology, SC-134220), respectively.

Techniques: ChIP-sequencing, Binding Assay, RNA Sequencing