Fig 2: BAFF/BCL10 signaling in hepatocye cell proliferation. (A) The expression of BAFFR mRNA in human CL-48 hepatocytes was determined by q-Reverse Transcription Polymerase Chain Reaction (q-RT-PCR); commercialized human peripheral blood mononuclear cells (PBMC) cDNA was used as the positive control. (B) Left panel, human CL-48 hepatocytes were treated without (control) or with BAFF (1 ng/mL) for 1 h, and the expression of BCL10 was determined by immunofluorescence staining; BCL10 was identified as a green signal, and the nucleus was stained with DAPI (blue). Magnification, 400×. Right panel, the number of BCL10 positive cells was counted under high power field (HPF). n = 6. * p < 0.05, by Student’s t-test. (C) CL-48 cells were treated with control or with BCL10 siRNA for 24 h; the expression of BCL10 was determined by western blotting. Actin was used as the loading control. (D) CL-48 cells were treated with control or BCL10 siRNA for 24 h prior treatment with BAFF (1 ng/mL). At different time points, relative cell proliferation was determined by the Trypan blue exclusion assay. The data are shown as mean ± SD of three independent experiments. Comparisons were made between the BAFF and bcl10siRNA + BAFF groups. * p < 0.05, by Student’s t-test. (E) CL-48 cells were treated as indicated with BCL10 siRNA or BAFF, as described in (D). On day 3, the cell cycle phases were determined by propidium iodide staining and FACScan analysis. Populations of cells in the sub-G1, G1, S, and G2/M phases were analyzed and quantified while using the Cell Quest software, and the data represent the means ± SDs of three independent experiments. Between-group comparisons were performed as indicated. * p < 0.05, by Student’s t-test.

Fig 3: Dominant-negative CARD11 variants in the open, reQM conformation perturb the ability of the all-mutant reQM CARD11 oligomer to recruit MALT1(A) NF-?B luciferase reporter activity in CARD11-KO Jurkat T cells stably transduced with myc-WT CARD11-FLAGx2 or myc-reQM CARD11-FLAGx2 with or without CARD11 LOF mutations. Mean fold activation with SD is depicted relative to Jurkat T cells reconstituted with WT CARD11. A two-tailed unpaired Student's t test with Welch's correction resulted in the following p values for the reQM CARD11 mutants as compared with that observed for reQM CARD11: E57D-reQM, p = 0.0016; and R72Q-reQM, p = 0.0013.(B) Reconstituted cells were harvested for denatured co-immunoprecipitation with rabbit anti-Bcl10 and Western blotting for linear ubiquitin, Bcl10, and FLAG.(C and D) MALT1 protease activation was assessed by Western blot analysis for HOIL-1, CYLD, and FLAG. The HOIL-1 and CYLD C-terminal cleavage products (HOIL-1Cter and CYLDCter) are indicated by arrows.(E–G) Cells were harvested for the endogenous co-immunoprecipitation with mouse anti-Bcl10 (E) or rabbit anti-FLAG (F and G) and lysate inputs and immunoprepcipiates were probed by Western blotting with the indicated antibodies.(H) The myc-reQM CARD11 variants were transiently transfected in HEK293T cells with 500 ng FLAG-MALT1 and 300 ng untagged Bcl10 as indicated for co-immunoprecipitation assays. At 40–41 h, cells were lysed and co-immunoprecipitated with rabbit anti-FLAG followed by Western blotting for myc, FLAG, and Bcl10. The following concentrations of DNA were used to achieve roughly equivalent protein expression for the FLAG-MALT1 co-immunoprecipitation: 400 ng reQM, 375 ng E57D-reQM, and 700 ng R72Q-reQM.

Fig 4: CARD and LATCH domain mutants disrupt the Opening Step in the CARD11 signaling cycle(A) NF-?B luciferase reporter activity in CARD11-KO Jurkat T cells stably expressing myc-WT-FLAGx2 or myc-mutant-FLAGx2 CARD11 variants via retroviral transduction after stimulation with anti-CD3/anti-CD28 for 4 h. Mean fold activation with SD from one of two replicate experiments is represented. A two-tailed unpaired Student's t test with Welch's correction resulted in the following p values for the values of the CARD11 mutants obtained under stimulated conditions as compared with that observed for wild-type CARD11 under stimulated conditions: N25Y, p = 0.0252; E27K, p = 0.0405; C28F, p = 0.0399; R30W, p = 0.0388; K41M, p = 0.0399; Q55R, p = 0.0396; E57D, p = 0.0322 R72Q, p = 0.0406; K83M, p = 0.0407; and H129D, p = 0.0245.(B) CARD11-KO Jurkat T cells reconstituted with myc-WT-FLAGx2 or myc-mutant-FLAGx2 were stimulated with PMA/ionomycin for 30 min, followed by the denatured co-immunoprecipitation assay with rabbit anti-Bcl10 and Western blotting for linear ubiquitin and Bcl10.(C) Reconstituted cells were stimulated with anti-CD3/anti-CD28 cross-linking for 30 min followed by Western blotting for CYLD, HOIL-1, and the loading controls IKKa and FLAG. The CYLD and HOIL-1 C-terminal cleavage products (CYLDCter and HOIL-1Cter) are indicated by arrows.(D) Reconstituted cells were stimulated with PMA/ionomycin for 30 min, followed by the endogenous co-immunoprecipitation assay with rabbit anti-FLAG and Western blotting for HOIP, MALT1, Bcl10, and FLAG.(E and F) Constructs expressing CARD11 mutants in the constitutively open and active myc-tagged RE Quadruple Mutant (myc-reQM) context were transiently transfected in HEK293T cells with (E) 200 ng FLAG-Bcl10 or (F) 100 ng FLAG-HOIP for co-immunoprecipitation assays. At 40–41 h, cells were lysed and co-immunoprecipitated with rabbit anti-FLAG followed by Western blotting with anti-myc and anti-FLAG. The following concentrations of DNA were used to achieve roughly equivalent protein expression for the FLAG-Bcl10 co-immunoprecipitation: 400 ng reQM, 575 ng N25Y-reQM, 600 ng E27K-reQM, 600 ng C28F-reQM, 675 ng R30W-reQM, 575 ng K41M-reQM, 650 ng Q55R-reQM, 400 ng E57D-reQM, 700 ng R72Q-reQM, 400 ng K83M-reQM, and 450 ng H129D-reQM. The following concentrations of DNA were adjusted to achieve roughly equivalent protein levels for the FLAG-HOIP co-immunoprecipitation: 500 ng reQM in the left panel and 400 ng reQM in the right panel, 500 ng of each reQM mutant.(G) CARD11-KO Jurkat T cells stably reconstituted with myc-WT-FLAGx2 or myc-mutant-FLAGx2 were stimulated with anti-CD3/anti-CD28 for 30 min followed by Western blotting to detect p-JNK and JNK2.(H) Reconstituted CARD11-KO Jurkat T cells were stimulated with anti-CD3/anti-CD28 for 24 h and IL-2 production was measured by ELISA. Data are represented as mean pg/mL of IL-2 with SD for one representative experiment performed with three technical replicates. A two-tailed unpaired Student's t test with Welch's correction resulted in the following p values for the values of the CARD11 mutants obtained under stimulated conditions as compared with that observed for wild-type CARD11 under stimulated conditions: N25Y, p = 0.0027; E27K, p = 0.0024; C28F, p = 0.0024; R30W, p = 0.0024; K41M, p = 0.0024; Q55R, p = 0.0024; E57D, p = 0.0024 R72Q, p = 0.0024; K83M, p = 0.0024; and H129D, p = 0.0016.

Fig 5: Activation of NF-?B in severe asthmatic fibroblasts. NHBF and DHBF were cultured in DMEM complete medium post serum-starvation. (A) Under basal conditions, mRNA expression of NF-?B pathway members, TLR4, BCL10, MALT1, CARMA3, I?Ba, A20, RELA, IL-6 and IL-8, in NHBF and DHBF was analyzed by qRT-PCR and expressed as fold expression change relative to NHBF post normalization to housekeeping gene 18s rRNA. (B) Whole cell lysates were subjected to immunoblot analysis of Bcl10 protein levels. ß-actin was used as loading control. Data are represented as mean ± SEM from at least 3 unique donors in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 determined by unpaired two-tailed Student t-test.