Fig. 6: Co-culture with hCD40L-3T3 cells upregulates NF-κB RelB and BCLXL in DLBCL, and selectively upregulates NF-κB cRel and MCL1 through signaling crosstalk when NF-κB RelA is chronically active.

A Abundances of the heterodimers RelA:p50 (left) and cRel:p50 (right) bound to inhibiting NF-κΒ proteins (IκB), as simulated by computational modeling for basal canonical NF-κΒ activation state in RIVA, and high basal activation state in SUDHL8. See supplementary modeling description. B Computational modeling results showing the nuclear abundance of the indicated NF-κB dimers in a simulation with low basal nuclear RelA (RIVA, pink), and a simulation high basal nuclear RelA (SUDHL8, teal). The mean (line) and standard deviation (shaded region) of 25 cells is indicated. C Schematic demonstrating the mechanism by which NF-κB contributes to the induction of BCLXL in a cell line with normal basal signaling (RIVA) upon stimulation with CD40L (right). hCD40L-3T3 mediated activation of NIK results in p100 processing into p52, leading to nuclear translocation of RelB:p52 and increased expression of BCLXL. Gray = inactive pathways and low abundance proteins, color = active pathways and predominant complexes. D Schematic demonstrating how crosstalk emerges between CD40, NIK, cRel, and MCL1. Increase basal RelA results in increase p100, and IκBδ (left). hCD40L-3T3 mediated activation of NIK results in processing of IκBδ and release of cRel:p50, which translocates to the nucleus and potentially upregulates expression of MCL1 (right). Gray = inactive pathways and low abundance proteins, color = active pathways and predominant complexes. Quantification of immunofluorescence microscopy in RIVA (E) and SUDHL8 (F) cells showing nuclear to cytoplasmic ratio of RelA, RelB and cRel. Data post 24 h of monoculture and hCD40L-3T3co-culture is shown side by side. Single cells of a representative replicate are shown (left), with violin width indicating data density. Quantification of the nuclear to cytoplasmic ratio of RelA, RelB, and cRel is shown as bar graphs (right) with error bars displaying the mean ± standard deviation of three independent experiments (*P < 0.05, **P < 0.01; unpaired ttest). G Schematic of the co-culture system used, following 24 h of treatment with 0.0001–100 μΜ of the BCLXL inhibitor A1331852 ± 0.1 μM of the Bruton’s Kinase (BTK) inhibitor Ibrutinib. H Cell viability of SUDHL8 in response to 0.0001–100 μΜ of the BCLXL inhibitor A1331852 post a 24-h treatment in monoculture, post a 24-h hCD40L-3T3co-culture or post a 24-h hCD40L-3T3 co-culture with the addition of 0.1 μM of the BTK inhibitor, Ibrutinib. LC₅₀ values for each condition is shown (right) with error bars representing the mean ± standard deviation of three independent experiments, normalized to the untreated control (***P < 0.001 one-way ANOVA with Tukey’s comparisons test).