Fig 2: siRNA-mediated knockdown of CARD9 attenuates high glucose-induced CHOP expression in INS-1 832/13 cells. Panel A: Following transfection with Con-si or CARD9-si as described previously, INS-1 832/13 cells were treated with LG or HG for 24 hrs. Relative abundance of CHOP and CARD9 in the cell lysates were determined by western blotting. ß-Actin was used as the control for protein loading. A representative blot from 3 independent experiments is shown here. Panel B: Densitometric quantification of CHOP expression in 3 independent experiments is shown here. Data are expressed as fold change relative to LG Con-si (* p<0.05). (Comparisons: a: significant compared with LG Con-si; b: significant compared with HG Con-si; *p<0.05). Panel C: Data depicting degree of inhibition of HG-induced CHOP expression in CARD9 depleted cells is shown here (* p<0.05; n=3).

Fig 3: CARD9 knockdown does not appear to impact high glucose-induced Caspase-3 activation in INS-1 832/13 cells. Panel A: Following transfection with Con-si or CARD9-si, INS-1 832/13 cells were treated with LG or HG for 24 hrs. Relative abundance of cleaved Caspase-3 (an index for Caspase-3 activation) and CARD9 in the cell lysates was determined by western blotting. ß-Actin was used as the control for protein loading. A representative blot from 6 independent experiments is shown here. Panel B: Densitometric quantitation of cleaved caspase-3 in Panel A is depicted herein. The pooled data are expressed as fold change relative to LG Con-si (mean ± SEM; n=6). (Comparisons: a: significant compared with LG Con-si; b: significant compared with LG CARD9-si; *p<0.05).

Fig 4: Assembly of the CARD-BCL10-MALT1 (CBM) signalosome drives all-or-none activation of nuclear transcription factor-?B (NF-?B).(A) Schematic diagram of binary activation in a population of cells. Higher doses of stimulation increase the probability, but not the magnitude, of activation by each cell. (B) ß-Glucan stimulation of THP-1 WT and BCL10-KO monocytes transduced with an NF-?B transcriptional reporter. Monocytes were stimulated for 24 hr then measured for T-Sapphire expression via flow cytometry. The graph shows the mean ± standard deviation (SD) fraction of cells positive for NF-?B expression of three biological replicates. The inset shows the histogram of T-Sapphire expression indicating the distinct negative and positive populations of THP-1 WT cells upon stimulation with 10 µg/ml ß-glucan. (C) HEK293 WT and BCL10-KO cells were transduced with an NF-?B transcriptional reporter and stimulated with PMA for 24 hr then measured for T-Sapphire expression via flow cytometry. The inset shows the histogram of T-Sapphire expression indicating the distinct negative and positive populations of HEK293T WT cells upon stimulation with 5 ng/ml PMA. Shown are means ± SD of three biological replicates. (D) THP-1 BCL10-KO monocytes were transduced with a doxycycline-inducible BCL10-mEos3.2 construct. Cells were imaged for BCL10 expression 24 hr after Dox induction. Images show BCL10 puncta only in cells treated with ß-glucan for 16 hr (see also Figure 1—figure supplement 3). In contrast, BCL10 E53R does not form puncta regardless of stimulation. Scale bar: 10 µm. (E) Quantification of the number of THP-1 cells with BCL10 puncta after ß-glucan stimulation. Each dot represents the average of 4 independent experiments with more than 50 cells each. Shown are means ± SD. (F) Endogenous untagged CARD9 and BCL10 form high molecular weight species in THP-1 cells stimulated with ß-glucan for 24 hr. After treatment, cells were lysed, and the protein extracts were resolved by size exclusion chromatography followed by capillary immunodetection. Figure 1—source data 1.Full image of protein immunodetection for CARD9 in lysates of THP-1 cells treated with ß-glucan. Figure 1—source data 2.Full image of protein immunodetection for CARD9 in lysates of untreated THP-1 cells. Figure 1—source data 3.Full image of protein immunodetection for BCL10 in lysates of THP-1 cells treated with ß-glucan. Figure 1—source data 4.Full image of protein immunodetection for BCL10 in lysates of untreated THP-1 cells.

Fig 5: Pathogenic mutations disrupt BCL10 template formation within CARD-CC multimers.(A) Schematic of the DAmFRET experiment used to test for nucleating interactions between CARD-BCL10-MALT1 (CBM) components. Artificial ‘seeds’ are created by expressing the putative nucleating protein as a fusion to the homo-oligomerizing module, µNS (purple). (B) DAmFRET plots of BCL10 (top) or CARDCARD (bottom) in the absence (left) or presence (right) of CARD9CARD seeds expressed in trans. For BCL10, the presence of seeds shifted all low-AmFRET cells to high-AmFRET, indicating that the protein had been supersaturated and is nucleated by CARD9CARD. In contrast, the seeds had no effect on CARD9CARD DAmFRET, suggesting that CARD9CARD only polymerizes at high concentrations and does not become supersaturated. The vertical line denotes the phase boundary for CARD9 CARD polymerization. (C) Graph of spline fits of AmFRET values for CARD9CARD mutants. All of the pathogenic mutations within the CARD domain disrupted polymer formation except for R70W, which stabilized polymers. (D) Effect of CARD9 mutations in the full-length protein context with autorepression eliminated by the I107E mutation. 293T cells transduced with the nuclear transcription factor-?B (NF-?B) reporter were transfected with the indicated CARD9-mCardinal constructs and analyzed for T-Sapphire expression at 48-hr post-transfection. T-Sapphire expression was normalized to that of cells transfected with plasmid expressing mCardinal alone. Shown are means ± standard deviation (SD). Analysis of variance (ANOVA) with post hoc Tukey test ***p < 0.0001. (E) Weibull fits to DAmFRET plots of BCL10 coexpressed with mutant CARD9 seeds, showing that only WT CARD9 nucleates BCL10 while none of the pathogenic mutants do so. (F) Images of yeast cells expressing the respective CARD9 seeds (red punctum) and BCL10-mEos3.1 (green). CARD9 WT seeds nucleate BCL10 polymers, resulting in a starburst-like structure. Conversely, CARD9-mutant R70W does not nucleate BCL10; BCL10 instead forms a single long filament that is detached from the ‘seed’, indicating that it polymerizes spontaneously in these cells. Scale bar: 5 µm.