Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: IRAK1 is not required for PEL survival. (A) IRAK1 Western blot of BCBL-1Cas9 cell lines showing complete knockout; loading control is β-actin. (B) Growth curves for BCBL-1Cas9 ΔIRAK1 clones obtained via trypan blue cell counting. Two ΔIRAK1 clones and an empty vector control were used in this experiment. (C) Representative images from colony formation assays of ΔIRAK1 BCBL-1Cas9 cells imaged at ×10 magnification. Cells were plated at a low cell density in 1% methylcellulose medium and grown for 3 weeks. (D) Quantification of colony formation in BCBL-1Cas9 ΔIRAK1 stable cell lines. Colony counts were obtained using ImageJ, and the square root of the number of colonies was plotted; n = 15. (E) Flanking cut-site PCR analysis using PerkinElmer LabChip GX-Touch. Primers were designed flanking the cut site. Image analysis revealed changes in band size of the KO versus that of WT cells.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Knock-Out, Clone Assay, Cell Counting, Plasmid Preparation, Stable Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: MYD88, IRAK1, and IRAK4 are dispensable in BC-1 cells. (A) MYD88 Western blot of BC-1Cas9 cell lines showing complete knockout; loading control is β-actin. (B) IRAK1 Western blot. (C) IRAK4 western blot. (D) Growth curves for BC-1Cas9 ΔMYD88 clones obtained via trypan blue cell counting. Two ΔMYD88 clones and an empty-vector WT control were used in this experiment. (E) Growth curves for BC-1Cas9 ΔIRAK1 clones. (F) Growth curves for BC-1Cas9 ΔIRAK4 clones. (G) Quantification of colony formation in BCBL-1Cas9 ΔMYD88 stable cell lines. Colony counts were obtained using ImageJ, and the square root of the number of colonies was plotted; n = 15. (H) Quantification of colony formation in BCBL-1Cas9 ΔIRAK1 stable cell lines. (I) Quantification of colony formation in BCBL-1Cas9 ΔIRAK4 stable cell lines.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Knock-Out, Clone Assay, Cell Counting, Plasmid Preparation, Stable Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: NF-κB activation by IL-1β is not functional in ΔMYD88 clones. (A) A Western blot for phospho-NF-κB and the IRAK pathway proteins IRAK1, IRAK4 and MYD88 in WT and ΔMYD88 BCBL-1Cas9 cells 15 min post IL-1β stimulation (1 ng/μl IL-1β). (B) Quantification of luciferase production using an NF-κB reporter assays system. Two ΔMYD88 clones and WT BCBL-1Cas9 cells were stimulated with 1 ng/μl IL-1β, or mock PBS for 24 h h following transfection, and luciferase values measured 6 h h post stimulation. Results are fold change over mock. (C) Two ΔMYD88 clones and WT BCBL-1Cas9 cells were stimulated with TNF-α (1 ng/ml), and the response was compared to mock using the same procedure as in panel B.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Activation Assay, Functional Assay, Clone Assay, Western Blot, Luciferase, Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: Complementation of IRAK1 restores signaling function in KO cells. (A) Western blot in WT BCBL-1Cas9 cells showing expression of Myc-tagged IRAK1 in BCBL-1Cas9 cells. (B) IRAK expression plasmids were conucleofected with an NF-κB reporter-driven luciferase plasmid into WT or ΔIRAK1 BCBL-1Cas9 cells. Cells were stimulated with IL-1β or PBS (mock), and luciferase values were measured 6 h poststimulation. Shown are relative activities adjusted across multiple biological replicates and scales as fraction of maximal response on a log10 scale. (C) IRAK expression plasmids were conucleofected with an NF-κB reporter-driven luciferase plasmid into WT, ΔIRAK1, ΔIRAK4, or ΔMYD88 BCBL-1Cas9 cells. Cells were stimulated with IL-1β, TNF-α, or PBS (mock), and luciferase values were measured 6 h poststimulation. Shown are relative activities adjusted across multiple biological replicates and scales as fraction of maximal response on a log10 scale.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Expressing, Luciferase, Plasmid Preparation

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: Comparison of in vitro and in culture IRAK inhibitor activity. (A) EC50 curves (growth) for three commercially available IRAK inhibitors. Fraction of response is shown on the vertical axis and concentration (in μM) on the horizontal axis. Inh1 (CAS no. 1042224-63-4), Inh4 (CAS no. 1012104-68-5), and Inh1-4 (CAS no. 509093-47-4). The EC50 value on each plot is the average from four experiments. (B) Quantification of luciferase production in cells transfected with an NF-κB-driven luciferase plasmid, incubated with inhibitor, a nd stimulated with 1 ng/μl IL-1β. Luciferase values were measured 6 h poststimulation. All values are fold change over that with mock PBS stimulation on the vertical axis and inhibitor concentration (in μM) on the horizontal axis. (C) A DiscoverX KINOMEscan analysis for each IRAK inhibitor at 250 nM. Purple or blue dots and represent IRAK4 or IRAK1 kinase, respectively. Size of the circle is proportional to percent activity inhibited by the inhibitors.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: In Vitro, Activity Assay, Concentration Assay, Luciferase, Transfection, Plasmid Preparation, Incubation

Journal: Frontiers in Oncology

Article Title: IRAK2, an IL1R/TLR Immune Mediator, Enhances Radiosensitivity via Modulating Caspase 8/3-Mediated Apoptosis in Oral Squamous Cell Carcinoma

doi: 10.3389/fonc.2021.647175

Figure Lengend Snippet: Higher IRAK2 expression was associated with a higher radiosensitivity in the context of parental (i.e., OML1) and radioresistant (i.e., OML1-R) OSCC cells. (A) After exposure to 0, 4, and 10 Gy IR, colony-formation assay confirmed that OML1-R cells were relatively radioresistant when compared with parental OML1 cells. (B) Venn diagram showed the number of genes with apparent expression change before and after irradiation in OML1 and OML1-R cells (left). Bar graphs displayed 19 genes were up-regulated in OML1 cells, using a filter criterion at least 1.5-fold change with P < 0.05. By setting a threshold of RPKM>2, we identified eight reliable transcripts that were largely differentially expressed between the OML1 and OML1-R cells. The graph showed relative fold change in gene expression: control versus IR-treated cells (right). (C) The RPKM value of IRAK2 expression was plotted for OML1 and OML1-R cells treated with 4 Gy. (D) qPCR and (E) Western blotting revealed that IRAK2 expression, including mRNA and protein levels, were pronouncedly elevated in parental OML1, but not OML1-R cells. Densitometry-derived values (bottom) were normalized with the control set as 1. β-actin served as the loading control for normalization.

Article Snippet: The plasmid for human IRAK2 (Myc-DDK-tagged) ectopic expression was purchased from Origene (Rockville, MD).

Techniques: Expressing, Colony Assay, Irradiation, Western Blot, Derivative Assay

Journal: Frontiers in Oncology

Article Title: IRAK2, an IL1R/TLR Immune Mediator, Enhances Radiosensitivity via Modulating Caspase 8/3-Mediated Apoptosis in Oral Squamous Cell Carcinoma

doi: 10.3389/fonc.2021.647175

Figure Lengend Snippet: Overexpression of IRAK2 restored radiosensitivity via enhancing radiation-induced apoptosis in OML1-R cells. (A) Colony formation assay showed that IRAK2-overexpressed OML1-R cells restored their radiosensitivity when compared with that of control OML1-R cells (P = 0.0100). (B) Apoptosis-specific flow cytometry represented that overexpression of IRAK2 significantly enhanced apoptosis in OML1-R cells before (52.28% vs. 0.62%) and after (63.50% vs. 0.81%) 4-Gy IR. The histogram on the right represent Annexin V-positive staining enrichment. (C) In OML1-R cells, protein levels of apoptosis-related factors, i.e., cleaved caspase-8, cleaved caspase-3, CHOP, and p65-NF-κB, were elevated by the overexpression of IRAK2, especially after 4-Gy IR (Western blotting, 72 hours after IR). (D) Protein levels of IRAK2, cleaved caspase-8 and cleaved caspase-3 were analyzed for OML1-R cells treated with an IRAK2 overexpression followed by the pretreatment with caspase-8 inhibitors (50 mM Z-IETD-FMK) for 1 hour. Densitometry-derived values (bottom) were normalized with the control set as 1. β-actin served as the loading control for normalization.

Article Snippet: The plasmid for human IRAK2 (Myc-DDK-tagged) ectopic expression was purchased from Origene (Rockville, MD).

Techniques: Over Expression, Colony Assay, Flow Cytometry, Staining, Western Blot, Derivative Assay

Journal: Frontiers in Oncology

Article Title: IRAK2, an IL1R/TLR Immune Mediator, Enhances Radiosensitivity via Modulating Caspase 8/3-Mediated Apoptosis in Oral Squamous Cell Carcinoma

doi: 10.3389/fonc.2021.647175

Figure Lengend Snippet: The knockdown of IRAK2 promoted resistance to IR-induced apoptosis in OML1 and SCC25 cells. (A) Endogenous IRAK2 expression in different OSCC cell lines, showing higher expressions of IRAK2 in OML1 and SCC25 than that of OML1-R and SCC4 cells. The values under bands represented the relative density that normalized to β-actin. (B) When compared with their control cells, post-irradiation colony formation rates were increased in IRAK2-knockdown OML1 (P = 0.0009) and SCC25 (P = 0.0577) cells. (C) Effect of radiation, IRAK2 shRNA or both on cell apoptosis in OML1 and SCC25 cell lines. Flow cytometry analysis using Annexin V and 7-AAD staining was performed to detect apoptotic cells. (D) IRAK2-shRNA transfection decreased the expressions of cleaved caspase-8 and caspase-3 in OML1 (left) and SCC25 (right) cancer cells. Densitometry-derived values (bottom) were normalized with the control set as 1. β-actin served as the loading control for normalization.

Article Snippet: The plasmid for human IRAK2 (Myc-DDK-tagged) ectopic expression was purchased from Origene (Rockville, MD).

Techniques: Expressing, Irradiation, shRNA, Flow Cytometry, Staining, Transfection, Derivative Assay

Journal: Frontiers in Oncology

Article Title: IRAK2, an IL1R/TLR Immune Mediator, Enhances Radiosensitivity via Modulating Caspase 8/3-Mediated Apoptosis in Oral Squamous Cell Carcinoma

doi: 10.3389/fonc.2021.647175

Figure Lengend Snippet: IRAK2 overexpression decreased OML1-R-generated in vivo tumor growth and enhanced radiosensitivity in the mice xenograft model. (A) In the mice xenograft model, IRAK2-overexpressed OML1-R-generated tumors had a relatively lower tumor growth rate than that of control OML1-R-generated tumors (P < 0.001). (B) Schema of cell injection and radiation treatments (upper panel). Since the 40th day after cancer cell injection, control and IRAK2-overexpressed mice were treated with RT per 4 days (i.e., a fraction size of 5 Gy by ten fractions to an accumulative dose of 50 Gy). The IRAK2-overexpressed mice had a smaller tumor volume than that of control mice at the time of radiotherapy (P = 0.0310; lower panel). (C) The expression of IRAK2, cleaved caspase-8, and cleaved caspase-3 in tumor tissues from control and IRAK2-overexpressed mice after radiotherapy were detected by immunohistochemical staining. (D) Immunoblotting analysis of the indicated proteins in lung tissues from control and IRAK2-overexpressed mice after irradiation. Densitometry-derived values (bottom) were normalized with the control set as 1. β-actin served as the loading control for normalization. Data were presented as mean ± SD. ‘***’ represented P < 0.001. All experiments were performed in triplicate.

Article Snippet: The plasmid for human IRAK2 (Myc-DDK-tagged) ectopic expression was purchased from Origene (Rockville, MD).

Techniques: Over Expression, Generated, In Vivo, Injection, Expressing, Immunohistochemical staining, Staining, Western Blot, Irradiation, Derivative Assay

Journal: Frontiers in Oncology

Article Title: IRAK2, an IL1R/TLR Immune Mediator, Enhances Radiosensitivity via Modulating Caspase 8/3-Mediated Apoptosis in Oral Squamous Cell Carcinoma

doi: 10.3389/fonc.2021.647175

Figure Lengend Snippet: Patient characteristics of 41 oral cancer patients according to the expression level of IRAK2.

Article Snippet: The plasmid for human IRAK2 (Myc-DDK-tagged) ectopic expression was purchased from Origene (Rockville, MD).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: IRAK2, an IL1R/TLR Immune Mediator, Enhances Radiosensitivity via Modulating Caspase 8/3-Mediated Apoptosis in Oral Squamous Cell Carcinoma

doi: 10.3389/fonc.2021.647175

Figure Lengend Snippet: After postoperative radiotherapy, OSCC patients with higher IRAK2 expressions showed better local recurrence-free survival (LRFS) than those with lower IRAK2 expressions. (A) Representative micrographs demonstrated the immunohistochemical (IHC) scores of IRAK2 expression in oral squamous cell carcinoma. (magnification, x200). (B) Kaplan-Meier survival curves represented that patients with higher IRAK2 expressions (i.e., >110) seemly demonstrated better 5-year LRFS than that of patients with lower expressions (i.e., 87.4% vs. 60.0%; P = 0.055, a statistical trend). (C) Cox proportional hazard regression confirmed this observation (univariate HR 0.25; 95% CI, 0.054 - 1.166; P = 0.055, a statistical trend). (D) Remarkably, after multivariable analysis, the two groups showed a statistically significant difference in terms of post-irradiation LRFS (multivariate HR 0.11; 95% CI, 0.016 - 0.760; P = 0.025). (E) Forest plot of multivariate analysis, depending on the Panel (D) Note that seven factors were used for multivariate analysis of LRFS (Panel C): age, gender, pathological stage, RT dose, chemotherapy, the status of surgical margin, and the expression level of IRAK2.

Article Snippet: The plasmid for human IRAK2 (Myc-DDK-tagged) ectopic expression was purchased from Origene (Rockville, MD).

Techniques: Immunohistochemical staining, Expressing, Irradiation

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: IRAK1 is not required for PEL survival. (A) IRAK1 Western blot of BCBL-1Cas9 cell lines showing complete knockout; loading control is β-actin. (B) Growth curves for BCBL-1Cas9 ΔIRAK1 clones obtained via trypan blue cell counting. Two ΔIRAK1 clones and an empty vector control were used in this experiment. (C) Representative images from colony formation assays of ΔIRAK1 BCBL-1Cas9 cells imaged at ×10 magnification. Cells were plated at a low cell density in 1% methylcellulose medium and grown for 3 weeks. (D) Quantification of colony formation in BCBL-1Cas9 ΔIRAK1 stable cell lines. Colony counts were obtained using ImageJ, and the square root of the number of colonies was plotted; n = 15. (E) Flanking cut-site PCR analysis using PerkinElmer LabChip GX-Touch. Primers were designed flanking the cut site. Image analysis revealed changes in band size of the KO versus that of WT cells.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Knock-Out, Clone Assay, Cell Counting, Plasmid Preparation, Stable Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: MYD88, IRAK1, and IRAK4 are dispensable in BC-1 cells. (A) MYD88 Western blot of BC-1Cas9 cell lines showing complete knockout; loading control is β-actin. (B) IRAK1 Western blot. (C) IRAK4 western blot. (D) Growth curves for BC-1Cas9 ΔMYD88 clones obtained via trypan blue cell counting. Two ΔMYD88 clones and an empty-vector WT control were used in this experiment. (E) Growth curves for BC-1Cas9 ΔIRAK1 clones. (F) Growth curves for BC-1Cas9 ΔIRAK4 clones. (G) Quantification of colony formation in BCBL-1Cas9 ΔMYD88 stable cell lines. Colony counts were obtained using ImageJ, and the square root of the number of colonies was plotted; n = 15. (H) Quantification of colony formation in BCBL-1Cas9 ΔIRAK1 stable cell lines. (I) Quantification of colony formation in BCBL-1Cas9 ΔIRAK4 stable cell lines.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Knock-Out, Clone Assay, Cell Counting, Plasmid Preparation, Stable Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: NF-κB activation by IL-1β is not functional in ΔMYD88 clones. (A) A Western blot for phospho-NF-κB and the IRAK pathway proteins IRAK1, IRAK4 and MYD88 in WT and ΔMYD88 BCBL-1Cas9 cells 15 min post IL-1β stimulation (1 ng/μl IL-1β). (B) Quantification of luciferase production using an NF-κB reporter assays system. Two ΔMYD88 clones and WT BCBL-1Cas9 cells were stimulated with 1 ng/μl IL-1β, or mock PBS for 24 h h following transfection, and luciferase values measured 6 h h post stimulation. Results are fold change over mock. (C) Two ΔMYD88 clones and WT BCBL-1Cas9 cells were stimulated with TNF-α (1 ng/ml), and the response was compared to mock using the same procedure as in panel B.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Activation Assay, Functional Assay, Clone Assay, Western Blot, Luciferase, Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: Complementation of IRAK1 restores signaling function in KO cells. (A) Western blot in WT BCBL-1Cas9 cells showing expression of Myc-tagged IRAK1 in BCBL-1Cas9 cells. (B) IRAK expression plasmids were conucleofected with an NF-κB reporter-driven luciferase plasmid into WT or ΔIRAK1 BCBL-1Cas9 cells. Cells were stimulated with IL-1β or PBS (mock), and luciferase values were measured 6 h poststimulation. Shown are relative activities adjusted across multiple biological replicates and scales as fraction of maximal response on a log10 scale. (C) IRAK expression plasmids were conucleofected with an NF-κB reporter-driven luciferase plasmid into WT, ΔIRAK1, ΔIRAK4, or ΔMYD88 BCBL-1Cas9 cells. Cells were stimulated with IL-1β, TNF-α, or PBS (mock), and luciferase values were measured 6 h poststimulation. Shown are relative activities adjusted across multiple biological replicates and scales as fraction of maximal response on a log10 scale.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Expressing, Luciferase, Plasmid Preparation

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: Comparison of in vitro and in culture IRAK inhibitor activity. (A) EC50 curves (growth) for three commercially available IRAK inhibitors. Fraction of response is shown on the vertical axis and concentration (in μM) on the horizontal axis. Inh1 (CAS no. 1042224-63-4), Inh4 (CAS no. 1012104-68-5), and Inh1-4 (CAS no. 509093-47-4). The EC50 value on each plot is the average from four experiments. (B) Quantification of luciferase production in cells transfected with an NF-κB-driven luciferase plasmid, incubated with inhibitor, a nd stimulated with 1 ng/μl IL-1β. Luciferase values were measured 6 h poststimulation. All values are fold change over that with mock PBS stimulation on the vertical axis and inhibitor concentration (in μM) on the horizontal axis. (C) A DiscoverX KINOMEscan analysis for each IRAK inhibitor at 250 nM. Purple or blue dots and represent IRAK4 or IRAK1 kinase, respectively. Size of the circle is proportional to percent activity inhibited by the inhibitors.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: In Vitro, Activity Assay, Concentration Assay, Luciferase, Transfection, Plasmid Preparation, Incubation

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: IRAK1 is not required for PEL survival. (A) IRAK1 Western blot of BCBL-1Cas9 cell lines showing complete knockout; loading control is β-actin. (B) Growth curves for BCBL-1Cas9 ΔIRAK1 clones obtained via trypan blue cell counting. Two ΔIRAK1 clones and an empty vector control were used in this experiment. (C) Representative images from colony formation assays of ΔIRAK1 BCBL-1Cas9 cells imaged at ×10 magnification. Cells were plated at a low cell density in 1% methylcellulose medium and grown for 3 weeks. (D) Quantification of colony formation in BCBL-1Cas9 ΔIRAK1 stable cell lines. Colony counts were obtained using ImageJ, and the square root of the number of colonies was plotted; n = 15. (E) Flanking cut-site PCR analysis using PerkinElmer LabChip GX-Touch. Primers were designed flanking the cut site. Image analysis revealed changes in band size of the KO versus that of WT cells.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Knock-Out, Clone Assay, Cell Counting, Plasmid Preparation, Stable Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: MYD88, IRAK1, and IRAK4 are dispensable in BC-1 cells. (A) MYD88 Western blot of BC-1Cas9 cell lines showing complete knockout; loading control is β-actin. (B) IRAK1 Western blot. (C) IRAK4 western blot. (D) Growth curves for BC-1Cas9 ΔMYD88 clones obtained via trypan blue cell counting. Two ΔMYD88 clones and an empty-vector WT control were used in this experiment. (E) Growth curves for BC-1Cas9 ΔIRAK1 clones. (F) Growth curves for BC-1Cas9 ΔIRAK4 clones. (G) Quantification of colony formation in BCBL-1Cas9 ΔMYD88 stable cell lines. Colony counts were obtained using ImageJ, and the square root of the number of colonies was plotted; n = 15. (H) Quantification of colony formation in BCBL-1Cas9 ΔIRAK1 stable cell lines. (I) Quantification of colony formation in BCBL-1Cas9 ΔIRAK4 stable cell lines.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Knock-Out, Clone Assay, Cell Counting, Plasmid Preparation, Stable Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: NF-κB activation by IL-1β is not functional in ΔMYD88 clones. (A) A Western blot for phospho-NF-κB and the IRAK pathway proteins IRAK1, IRAK4 and MYD88 in WT and ΔMYD88 BCBL-1Cas9 cells 15 min post IL-1β stimulation (1 ng/μl IL-1β). (B) Quantification of luciferase production using an NF-κB reporter assays system. Two ΔMYD88 clones and WT BCBL-1Cas9 cells were stimulated with 1 ng/μl IL-1β, or mock PBS for 24 h h following transfection, and luciferase values measured 6 h h post stimulation. Results are fold change over mock. (C) Two ΔMYD88 clones and WT BCBL-1Cas9 cells were stimulated with TNF-α (1 ng/ml), and the response was compared to mock using the same procedure as in panel B.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Activation Assay, Functional Assay, Clone Assay, Western Blot, Luciferase, Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: Complementation of IRAK1 restores signaling function in KO cells. (A) Western blot in WT BCBL-1Cas9 cells showing expression of Myc-tagged IRAK1 in BCBL-1Cas9 cells. (B) IRAK expression plasmids were conucleofected with an NF-κB reporter-driven luciferase plasmid into WT or ΔIRAK1 BCBL-1Cas9 cells. Cells were stimulated with IL-1β or PBS (mock), and luciferase values were measured 6 h poststimulation. Shown are relative activities adjusted across multiple biological replicates and scales as fraction of maximal response on a log10 scale. (C) IRAK expression plasmids were conucleofected with an NF-κB reporter-driven luciferase plasmid into WT, ΔIRAK1, ΔIRAK4, or ΔMYD88 BCBL-1Cas9 cells. Cells were stimulated with IL-1β, TNF-α, or PBS (mock), and luciferase values were measured 6 h poststimulation. Shown are relative activities adjusted across multiple biological replicates and scales as fraction of maximal response on a log10 scale.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Expressing, Luciferase, Plasmid Preparation

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: Comparison of in vitro and in culture IRAK inhibitor activity. (A) EC50 curves (growth) for three commercially available IRAK inhibitors. Fraction of response is shown on the vertical axis and concentration (in μM) on the horizontal axis. Inh1 (CAS no. 1042224-63-4), Inh4 (CAS no. 1012104-68-5), and Inh1-4 (CAS no. 509093-47-4). The EC50 value on each plot is the average from four experiments. (B) Quantification of luciferase production in cells transfected with an NF-κB-driven luciferase plasmid, incubated with inhibitor, a nd stimulated with 1 ng/μl IL-1β. Luciferase values were measured 6 h poststimulation. All values are fold change over that with mock PBS stimulation on the vertical axis and inhibitor concentration (in μM) on the horizontal axis. (C) A DiscoverX KINOMEscan analysis for each IRAK inhibitor at 250 nM. Purple or blue dots and represent IRAK4 or IRAK1 kinase, respectively. Size of the circle is proportional to percent activity inhibited by the inhibitors.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: In Vitro, Activity Assay, Concentration Assay, Luciferase, Transfection, Plasmid Preparation, Incubation