Fig 1: Conditional BCL6 knock-out in SU-DHL-4 induces gene perturbations similar to BCL6 degradation.RNA-seq analysis was performed to compare gene expression after BCL6 knock-out and compound-induced degradation. (A) Volcano plot visualizing log2-scaled fold changes (x-axis) induced by either BI-3802 mediated degradation (compared to DMSO treatment) or BCL6 knock-out (compared to control sgRNA treatment) versus statistical significances (-log10 of the adj. p-value on the y-axis). Significantly deregulated genes (adj. p-value = 0.01, fold change = 3) are depicted in blue and red for repressed and induced genes, respectively. (B) Correlation of changes in gene expression induced by BCL6 knock-out (x-axis) or BI-3802 mediated degradation (y-axis). Genes near the dotted lines show comparable expression modulation in the BI-3802 treated data versus the BCL6 knock-out data set. Blue lines show linear regressions of the actual fold-change values. The goodness-of-fit of the linear regressions are shown by the r2 value in the graphs. (C) Gene set enrichment analysis (selected terms, FDR = 0.1) reflecting genes set that are enriched/depleted for genes modulated by BCL6 knock-out or BI-3802 mediated degradation. The normalized enrichment score (NES) is color-coded in the heatmap. Negative values indicate gene sets that are significantly enriched for genes that are down-regulated upon BCL6 knock-out or BI-3802 treatment as shown in Supplementary Figure 4C (cell cycle). (D) Venn diagram indicating the overlap of genes after BCL6 degradation and BCL6 knock-out in SU-DHL-4 cells at the indicated time points of BI-3802 and DOX treatment.
Fig 2: Conditional BCL6 knock-out in SU-DHL-4 in vitro induces anti-proliferative effects.Long-term proliferation assays with (A) BCL6 sgRNA and (B) negative control infected SU-DHL-4 Cas9 cells after DOX induction. For this assay cells were kept at constant concentrations of 3 different DOX concentrations as indicated and split to 200,000 cells per ml every 3–4 days. Split rates were multiplied to derive growth curves. BCL6 protein expression was determined at the indicated time points after DOX induction (100 ng/ml) in (C) BCL6 and (D) control sgRNA SU-DHL-4 Cas9 cells after immunohistochemical staining of cell pellets. (E) Caspase 3/7 activity and (F) cell cycle analysis after 4–10 days DOX treatment were investigated in SU-DHL-4 BCL6 sgRNA transduced cells. Data are shown as means ± SD of independent experiments (n = 2 – 8). ** p = 0.01; *** p = 0.001.
Fig 3: Depletion of BCL6 knock-out DLBCL cells in bulk assays.A time course CRISPR depletion experiment, following the depletion kinetics of GFP+ cells (Cas9 and sgRNA expressing) relative to the GFP- cells (Cas9-expressing) in the DLBCL cell lines OCI-Ly1, KARPAS-422, SU-DHL-4 and Toledo and the breast cancer cell line MCF-7. POLR2A serves as a core essential control gene. NegCtrl depicts a non-targeting control and BCL6 sgRNAs 1–7 are BCL6 specific sgRNAs. Data are shown as relative GFP expression to the pos Ctrl sgRNA POLR2A on day 17 post infection.
Fig 4: Localisation and expression of BCL6 and HMGB1. (A) Representative images of BCL6 positive cells (brown staining) in the stroma in the vicinity of PanIN lesions are shown in the top two panels (both magnified ×100); two bottom images show inflammatory infiltrate with BCL6 immunoreactive cells in two PDAC cases (magnification ×100 and ×50, respectively). (B) HMGB1 nuclear expression (brown staining) was seen in all pancreatic compartments, including stromal immune infiltrate: top panels show PanIN-1 (left) and -2 (right) (magnification ×50, insert and second panel x100); bottom panels show PanIN-3 (left) and PDAC (right) (magnification ×100 and ×50, respectively).
Fig 5: BCL6 knock-out in a DLBCL xenograft induces tumor stasis.Tumor xenografts were established in C.B-17 SCID mice by subcutaneous injection of inducible SU-DHL-4 Cas9 BCL6 and control sgRNA cells. Mice were randomized to receive drinking water with DOX (2 mg/kg) plus 5% sucrose (DOX on) or 5% sucrose only (DOX off). (A) After 5 days DOX treatment tumors from four mice were harvested and analyzed for Cas9 GFP induction using flow cytometry. Cas9-GFP-induced cells are indicated in green, non-induced cells in red. (B–E) Tumor-bearing mice were treated with DOX for 8 days after which tumors from control and BCL6 knock-out tumors were harvested 17/20 days after start of DOX treatment, respectively. Tumor volumes from (B) BCL6 sgRNA tumors (n = 10 DOX off, n = 7 DOX on) and (C) control (n = 10 DOX off, n = 8 DOX on) were measured. * p < 0.05; *** p = 0.001. (D) Tumor BCL6 protein levels were determined using IHC analysis. Representative images of BCL6 IHC staining in SU-DHL-4 tumors are shown. Scale bars 100 µm. (E) Quantification of BCL6 positive cells in SU-DHL-4 BCL6 sgRNA tumor sections after vehicle (DOX off) and DOX treatment (5 days and 20 days after start of DOX treatment). Data are shown as means ± SD relative to DOX off (n = 4 – 10).
Supplier Page from MilliporeSigma for Anti-BCL6 antibody produced in rabbit