bjab b cell lymphoma lines  (DSMZ)

Journal: Molecular Therapy

Article Title: Development of RelB-targeting small-molecule inhibitors of non-canonical NF-κB signaling with antitumor efficacy

doi: 10.1016/j.ymthe.2025.01.048

Figure Lengend Snippet: RS47 blocks non-canonical NF-κB signaling pathway (A and B) Inhibitory effects of RS47 on the NF-κB (A) and BCL3 (B) luciferase reporter activity. (C and D) Biotinylated RS47 pull-down assay using protein lysate from HCT116 cells (C) or using purified RelB protein (D). (E) MST assay showed the affinity between RS47 and purified RelB protein. (F) Biotinylated RS47 pull-down assay using protein lysate from 293T cells overexpressing WT RelB or mutated RelB. (G) Representative IF image shows the co-localization of RelB and RS47. White arrows indicate co-localization site in nucleus. Scale bars, 20 μm. (H) Non-canonical NF-κB DNA-binding activity interrupted by RS47 was assessed by EMSA. (I) Heatmap of BAFF-inducible genes (FC > 2, p < 0.05) inhibited by RS47 in BJAB cells. (J) GSEA enrichment of differentially expressed genes centralized in non-canonical NF-κB signaling pathway. (K) The mRNA expression of Pim2 in BJAB cells and primary B cells. (L) Flow cytometry profiling of Annexin V/7-AAD-stained PDX cells. (M and N) The CCK-8 results of HCT116 cell knockdown (M) or overexpressed (N) RelB. Error bars indicate the SEM of repeated assays. Each experiment was repeated at least three times. Two-way ANOVA with Tukey correction for (A), (B), and (L). Unpaired two-tailed t test for (K), (M), and (N). ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Human colon cancer cell line HCT116, RKO, 231 and SW620, human colon normal cell CRL1459, B lymphoma cell line BJAB, U2932, and breast cancer cell line MDA-MB231 were purchased from ATCC.

Techniques: Luciferase, Activity Assay, Pull Down Assay, Purification, Binding Assay, Expressing, Flow Cytometry, Staining, CCK-8 Assay, Knockdown, Two Tailed Test

Journal: Molecular Therapy

Article Title: Development of RelB-targeting small-molecule inhibitors of non-canonical NF-κB signaling with antitumor efficacy

doi: 10.1016/j.ymthe.2025.01.048

Figure Lengend Snippet: RS47 has no inhibitory effect on the canonical NF-κB signaling pathway (A) Biotinylated RS47 pull-down assay using protein lysate from HCT116 cells. (B) The inhibitory effect of RS47 on canonical NF-κB DNA-binding activity was assessed by EMSA. (C and D) The expression of TNF mRNA in BJAB cells (C) and U2932 cells (D). (E) Cartoon representation of the crystal structure of RelA (green)/DNA complex (gray) and RelB (blue)/DNA complex (magenta). (F and G) Cartoon representation of RS47 (pink sticks) docked onto the crystal structure of RelA/DNA complex (F) and RelB/DNA complex (G). Error bars indicate the SEM of repeated assays. Each experiment from (A) to (D) was repeated at least three times. Unpaired two-tailed t test for (C). ns, not significant; ∗∗∗ p < 0.001.

Article Snippet: Human colon cancer cell line HCT116, RKO, 231 and SW620, human colon normal cell CRL1459, B lymphoma cell line BJAB, U2932, and breast cancer cell line MDA-MB231 were purchased from ATCC.

Techniques: Pull Down Assay, Binding Assay, Activity Assay, Expressing, Two Tailed Test

Journal: Molecular Therapy

Article Title: Development of RelB-targeting small-molecule inhibitors of non-canonical NF-κB signaling with antitumor efficacy

doi: 10.1016/j.ymthe.2025.01.048

Figure Lengend Snippet: RS47 induces B lymphoma cells apoptosis (A) Flow cytometry profiling of Annexin V/7-AAD-stained BJAB cells (left). Summary of the results (right). (B) Flow cytometry profiling of Annexin V/7-AAD-stained U2932 cells (left). Summary of the result (right). (C) Flow cytometry profiling of Annexin V/7-AAD-stained PDX cells in co-culture experiments. (D) Summary of the result in (C). (E) Flow cytometry profiling of Annexin V/7-AAD-stained BMSCs in co-culture experiments. (F) Summary of the result in (E). (G) The sphere number of BJAB cells. Scale bars, 100 μm. (H) Summary of the result in (G). Error bars indicate the SEM of repeated assays. Each experiment was repeated at least three times. Unpaired two-tailed t test for (A), (B), (D), (F), and (H). ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Human colon cancer cell line HCT116, RKO, 231 and SW620, human colon normal cell CRL1459, B lymphoma cell line BJAB, U2932, and breast cancer cell line MDA-MB231 were purchased from ATCC.

Techniques: Flow Cytometry, Staining, Co-Culture Assay, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: The Overexpression of Collagen Receptor DDR1 is Associated With Chromosome Instability and Aneuploidy in Diffuse Large B‐Cell Lymphoma

doi: 10.1111/jcmm.70318

Figure Lengend Snippet: Activated DDR1 downregulates expression of the mitotic kinesin, CENPE . (A) Upper panels show the significant overlap between genes upregulated by DDR1 in normal GC B cells and those upregulated in either GCB‐ or ABC‐DLBCL compared with normal GC B cells (total genes on both platforms = 26,438). The lower panel shows GO analysis of genes upregulated by DDR1 in normal GC B cells and also upregulated in both GCB and ABC‐DLBCL. Open bars indicate the percentage of genes present in the GO term that would be expected to be observed in the regulated gene list by chance. Shaded bars indicate the percentage of genes in the regulated list with a given GO term that were actually observed. (B) RT‐qPCR confirming that collagen treatment of DDR1‐expressing GC B cells significantly reduced the mRNA levels of CENPE . Shown are triplicate data on tonsillar GC B cells isolated from three different donors (T20, T21 and T42). (C) Compared to empty vector (EV) control, the addition of collagen significantly reduced CENPE mRNA expression in DDR1‐expressing B‐cell lines, BJAB (maximally after 6 h of stimulation) and DG75 (maximally after 2 h of stimulation). Data are representative of three separate biological replicates. (D) Activation of an ectopically expressed DDR1 reduced CENPE protein expression in both BJAB and DG75 cells. An antibody specific for DDR1 phosphorylated on Tyr792 was used to confirm activation of DDR1. As expected, the activation of DDR1 also reduced total DDR1 levels. β‐Actin was used as a loading control. Data shown are representative of three separate biological replicates. (E) A constitutively activated DDR1 construct also downregulated CENPE protein expression. Shown here are the mean fluorescence intensities for DDR1 and CENPE in DG75 cells transfected with the constitutively active DDR1 gene. Cells expressing DDR1 were significantly more likely to lack CENPE protein expression than were untransfected cells in the same population ( p < 0.0001). (F) Immunohistochemistry showing the downregulation of CENPE protein in DLBCL tumour cells. Top left panel shows strong expression of CENPE protein in a normal germinal centre (GC) of tonsil. Original magnification ×200. Remaining panels show examples of staining in a case in which CENPE was not detected in tumour cells (top right), a case with weak expression in tumour cells (bottom left) and a case showing strong staining (bottom right). Original magnification ×400. Black arrows show tumour cells. Red arrows indicate nonmalignant cells that are positive for CENPE.

Article Snippet: DG75 and BJAB B‐cell lymphoma lines (DSMZ, Braunschweig, Germany) were cultivated in RPMI 1640 (Gibco; Life Technologies Ltd., Paisley, UK) culture media, with 10% FCS and 1% penicillin–streptomycin (Gibco).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Plasmid Preparation, Control, Activation Assay, Construct, Fluorescence, Transfection, Immunohistochemistry, Staining