Tissue microarray based immunohistochemical analysis of p-IκB expression in DLBCL patients: DLBCL array spot showing high expression of p-IκB (A) and low expression of p-IκB (B).

<p>20×/0.70 objective on an Olympus BX 51 microscope. (Olympus America Inc, Center Valley, PA, USA. with the inset showing a 40×0.85 aperture magnified view of the same. (C). Impact of p-IκB expression on prognosis in ABC patients. In patients with activated B cell phenotype(ABC) subgroup, p-IκB over expression showed a poor overall survival of 56.3% at 5 years as compared to 78.2% with low p-IκB expression (p = 0.0724).</p

TQ-induced up-regulation of DR5 does not play a part in TQ-induced apoptosis.

<p>(A) TQ treatment causes up-regulation of DR5 in ABC cells. HBL1 and RIVA cells were treated with 10 µM TQ for indicated time periods. After cell lysis, equal amounts of proteins were immuno-blotted with antibodies against DR5 and beta actin. (B) TQ-induced DR5 up-regulation is ROS dependent. HBL-1 cells were pre-treated with either 10 mM NAC or 80 µM z-VAD for 3 hours and subsequently treated with 10 µM TQ for 24 hours. Cells were lysed and equal amounts of proteins were immunoblotted with antibodies against DR5 and beta-actin. (C and D) TQ-induced apoptosis is not DR5 dependent. (C) HBL-1 and RIVA cells were either transfected with 50 and 100 nM siRNA, specific against DR5 or scrambled siRNA for 48 hours. Cells were then treated with 5 and 10 µM TQ for 24 hours, following which cells were stained with fluorescent-conjugated Annexin V/PI and analyzed by flow cytometry. Bar graph displays the mean +/− SD (standard deviation) of three independent experiments, * p<0.05, statistically significant (Students <i>t</i>-test). (D) HBL-1 and RIVA cells were either transfected with 50 and 100 nM siRNA, specific against DR5 or scrambled siRNA for 48 hours and treated with 5 and 10 µM TQ for 24 hours. Cells were lysed and equal amounts of proteins were immuno-blotted with antibodies against DR5, caspase-8, caspase-3, PARP and beta-actin. (E): Combination treatment with sub-optimal doses of TQ and TRAIL induce synergistic inhibition of cell viability and apoptosis in ABC cells. HBL-1 and RIVA cells were treated with either 1 µM TQ in the presence and absence of 1 ng TRAIL for 24 hours. Following treatment, cells were analyzed for cell viability by MTT assay as described in material and methods. Bar graph displays the mean +/− SD (standard deviation) of three independent experiments, * p<0.05, statistically significant (Students <i>t</i>-test). (F) HBL-1 and RIVA cells were treated with either 1 µM TQ in the presence and absence of 1 ng TRAIL for 24 hours. Following treatment, cells were stained with fluorescent-conjugated Annexin V/PI and analyzed by flow cytometry (G) HBL-1 cells were treated with either 1 µM TQ in the presence and absence of 1 ng TRAIL for 24 hours. Following treatment, cells were lysed and equal amounts of proteins were immuno-blotted with antibodies against caspase-8, caspase-3, PARP, and beta-actin.</p

Correlation of p-IκB expression with clinico-pathological parameters in ABC subtypes of DLBCL samples.

<p>^@IPI & Stage information was available only in 89 patients. ?GC vs. ABC Germinal Centre (GC) versus Activated B Cell (ABC) phenotype.^$Of the 201 Diffuse Large B cell lymphomas, p-IκB results were available in 151 cases and the remaining 50 spots were non informative. Analysis failure for these IHC markers was attributed to missing or non representative spots. Of these 151 TMA spots with available p-IκB data, range of non available spots for rest of the IHC markers ranged from 1 spot for XIAP to 18 spots for XIAP. The remaining cases were considered for correlation analysis.</p

Thymoquinone-induced generation of ROS in ABC cells.

<p>(<b>A</b>) <b>Thymoquinone increases ROS generation in ABC cells.</b> HBL-1 and RIVA cells were incubated in the absence or presence of 10 µM TQ for indicated time periods. After washing with PBS, cells were incubated with 10 µM H2DCFDA and incubated in the dark for 30 minutes at 37°C as described in material and method. Cells were washed in PBS, re-suspended in PBS and analyzed by flow cytometry. (<b>B</b>) <b>NAC prevents TQ-induced ROS release in ABC cells.</b> HBL-1 and RIVA cell lines were pre-treated with 10 mM NAC for 21hours followed by treatment with 10 µM TQ for various time periods. Cells were washed in PBS and incubated with 10 µM H2DCFDA and incubated in the dark for 30 minutes at 37°C. Cells were washed in PBS, re-suspended in PBS and analyzed by flow cytometry. Bar graph displays the mean +/− SD (standard deviation) of three independent experiments, * p<0.05, statistically significant (Students <i>t</i>-test). (<b>C</b>) (<b>C</b>) <b>NFκB activity is down-regulated by TQ as measured by Luciferase assay.</b> RIVA cells were transiently transfected with p65-LUC plasmid for 48 hours. After transfection, RIVA cells were divided into three groups; one group was left untreated as control, the second group was treated with 10 µM TQ for 24 hours and third group was pretreated with 10 mM NAC for 3 hours followed by treatment with 10 µM TQ for 24 hours. After 24 hours, cells were lysed in luciferase lysis buffer and luciferase activity was measured. pCDNA plasmid was used as transfection control and β-galactosidase was used to normalize the transfection efficiency. <b>(D)</b><b>NAC prevents TQ-induced down-regulation of targets of p65.</b> HBL-1 and RIVA cells were pre-treated with 10 mM NAC for three hours followed by treatment with 10 µM TQ for 24 hours. Cells were lysed and proteins were immunoblotted with antibodies against IκBα, Bcl-2 and Survivin.</p

TQ-induced mitochondrial signaling pathway in ABC cells.

<p>(A) TQ treatment causes Bax conformational changes in ABC cells. After treating with 10 µM TQ for indicated time periods, HBL-1 cells were lysed and immuno-precipitated with anti-Bax 6A7 antibody for detection of conformationally changed Bax protein. In addition, the total cell lysates were immuno-blotted with specific anti-Bax polyclonal antibody. (B) NAC prevents TQ-induced Bax conformational changes in ABC cells. HBL-1 cells were pre-treated with either, 10 mM NAC and 80 µM z-VAD/fmk for 3 hours and subsequently treated with 10 µM TQ for 8 hours. Cells were lysed and immunoprecipitated with anti-Bax 6A7 antibody and proteins were immunoblotted with Bax rabbit polyclonal antibody. (C) TQ treatment causes change in mitochondrial membrane potential in ABC cells. ABC cells were treated with and without 5 and 10 µM TQ for 24 hours. Live cells with intact mitochondrial membrane potential and dead cells with lost mitochondrial membrane potential was measured by JC-1 staining and analyzed by flow cytometry as described in Materials and Methods. Bar graph displays the mean +/− SD (standard deviation) of three independent experiments, * p<0.05, statistically significant (Students <i>t</i>-test). (D) TQ treatment causes release of cytochrome c from mitochondria into cytosole. HBL-1 and RIVA cells were treated with 5 and 10 µM TQ for 24 hours. Mitochondrial free cytosolic fractions were isolated and immunoblotted with antibody against cytochrome c and Beta-actin.</p

TQ-induced caspases dependent apoptosis in ABC cells.

<p>(A) TQ treatment causes activation and cleavage of caspases in ABC cells. HBL-1 and RIVA cells were treated with and without 5 and 10 µM TQ for 24 hours. Cells were lysed and equal amounts of proteins were immunoblotted with antibodies against caspase-9, caspase-3, PARP and Beta-actin. (B) TQ-induced caspases activation and cleavage is blocked by NAC and caspases inhibitor. HBL-1 cells were pretreated with either 10 mM NAC or 80 µM z-VAD for 3 hours and subsequently treated with 10 µM TQ for 24 hours. Cells were lysed and equal amounts of proteins were immunoblotted with antibodies against caspase-9, caspase-3 cleaved caspase-3, PARP and beta-actin. (C) TQ suppresses growth of ABC cells. ABC cell lines were incubated with 0–50 µM TQ for 24 hours. Cell viability was measured by MTT assays as described in Materials and Methods. The graph displays the mean +/− SD (standard deviation) of three independent experiments, * p<0.05, statistically significant (Students <i>t</i>-test). (D) TQ treatment induces apoptosis in ABC cell lines. ABC cells were treated with 5 and 10 µM TQ (as indicated) for 24 hours and cells were subsequently stained with flourescein-conjugated annexin-V and propidium iodide (PI) and analyzed by flow cytometry.</p

Down-regulation of NFκB pathway leads to inhibition of cell growth and induces apoptosis in PEL cell lines.

<p>(<b>A</b>) <b>Bay11-7085 suppresses growth of PEL cells.</b> PEL cell lines were incubated with 0–25 µM Bay11-7085 for 24 hours. Cell viability was measured by MTT assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039945#s2" target="_blank">Materials and Methods</a>. The graph displays the mean +/− SD (standard deviation) of three independent experiments, * p<0.05, statistically significant (Students <i>t</i>-test). <b>Bay11-7085 treatment induces apoptosis in PEL cells</b> (<b>B</b>) PEL cells were treated with 5 and 10 µM Bay11-7085 for 24 hours and apoptosis was measured by Live/Dead Assay. (<b>C</b>) PEL cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Thereafter, the cells were washed, fixed and stained with propidium iodide, and analyzed for DNA content by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039945#s2" target="_blank">“Materials and methods”</a>. (<b>D</b>) PEL cells were treated with 5 and 10 µM Bay11-7085 (as indicated) for 24 hours and cells were subsequently stained with flourescein-conjugated annexin-V and propidium iodide (PI) and analyzed by flow cytometry.</p

Bay11-7085 treatment of PEL cells activates mitochondrial apoptotic pathway in PEL cell lines.

<p>(<b>A</b>) <b>Bay11-7085-induced Bax activation in PEL cells.</b> After treating with 10 µM Bay11-7085 for indicated time periods, BC1 cells were lysed in 1% Chaps lysis buffer and subjected to immuno-precipitation with anti-Bax 6A7 antibody for detection of conformationally changed Bax protein. In addition, the total cell lysates were applied directly to SDS–PAGE, transferred to immobilon membrane and immuno-blotted with specific anti-Bax polyclonal antibody. (<b>B</b>) <b>Bay11-7085 treatment causes change in mitochondrial membrane potential in PEL cells.</b> PEL cells were treated with and without 5 and 10 µM Bay11-7085 for 24 hours. Live cells with intact mitochondrial membrane potential and dead cells with lost mitochondrial membrane potential was measured by JC-1 staining and analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039945#s2" target="_blank">Materials and Methods</a>. (<b>C</b>) <b>Bay11-7085 treatment causes release of cytochrome c from mitochondria into cytosole in PEL cells.</b> BC1 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Mitochondrial free cytosolic fractions and cytosolic extracts were isolated and immunoblotted with antibody against cytochrome c and Beta-actin. (<b>D</b>) <b>Bay11-7085 treatment causes down-regulation of IAPs in PEL cells.</b> BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against cIAP1 and cIAP2. Beat actin was used for equal loading.</p

Cross-talk between NFκB and PI3-kinase/AKT pathway in PEL cell lines.

<p>(<b>A</b>) <b>Bay11-7085 treatment inactivates AKT and its down-stream targets in PEL cells.</b> BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against p-AKT, p-FOXO1, p-GSK3, p-Bad and Beta-actin. (<b>B</b>) <b>Transcriptional knock down of p65 causes in-activation of AKT and its down-stream targets in PEL cells.</b> BC1 and BC3 cells were transfected with siRNA against p65 for 48 hours. Following transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against p-AKT, p-FOXO1, p-GSK3, p-Bad and Beta-actin. (<b>C</b>) <b>Transcriptional targeting of AKT causes in-activation of NFκB pathway.</b> BC1 cells were transfected with siRNA against AKT for 48 hours. Following transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against p-AKT, p-IκBα and Beta-actin.</p

Combination of NFκB and PI3-kinase/AKT inhibitors induce synergistic inhibition of cell viability and induction of apoptosis in PEL cell lines.

<p>(<b>A</b>) <b>Combination of sub-toxic doses of Bay11-7085 and LY294002 suppresses growth of PEL cells.</b> BC1 and BC3 cells were treated with either 1 µM Bay11-7085 or 5 µM LY294002 alone or in combination for 24 hours. Cell viability was measured by MTT assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039945#s2" target="_blank">Materials and Methods</a>. The graph displays the mean +/− SD (standard deviation) of three independent experiments, * p<0.05, statistically significant (Students <i>t</i>-test). (<b>B and C</b>) <b>Combination of Bay11-7085 and LY294002 induces efficient apoptosis in PEL cells.</b> BC1 and BC3 cells were treated with either 1 µM Bay11-7085 or 5 µM LY294002 alone or in combination for 24 hours. Following treatment, cells were stained with FITC conjugated annexin V/PI and cells were analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039945#s2" target="_blank">material and methods</a> (<b>B</b>). Bar graph denotes percentage apoptosis of three independent experiments (<b>C</b>). (<b>D</b>) <b>Combination of sub-toxic doses of Bay11-7085 and LY294002 induced apoptosis is caspase dependent.</b> BC1 cells were treated with either 1 µM Bay11-7085 or 5 µM LY294002 alone or in combination for 24 hours. Following treatment, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-9, caspase-3, cleaved caspase-3 and PARP. Beta-actin was used for equal loading.</p