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irak4  (MedChemExpress)


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    Structured Review

    MedChemExpress irak4
    (A) Schematic of UR241-2 design and synthesis. This figure illustrates the logarithm of partition coefficient (LogP), topological polar surface area (tPSA), calculated logarithm of partition coefficient (CLogP), and logarithm of solubility (LogS). (B) Computer-simulated docking analyses for UR241-2 and PF06650833 against IRAK1 and <t>IRAK4</t> using MedusaDock program. (C) The half maximal inhibitory concentration (IC 50 ) of UR241-2 for IRAK1 and IRAK4 measured by kinase profiling assay with protein suspension. UR241-2 underwent testing in 10-dose IC 50 duplicate mode, starting at 20 µM with 3-fold serial dilution. Reactions were carried out at 1 µM ATP. (D) The cellular IC 50 of UR241-2 for IRAK1 and IRAK4 measured by NanoBRET Target Engagement Assay in HEK293 cells. HEK293 cells were transiently transfected with 1 µg IRAK1 and IRAK4 NanoLuc Fusion Vectors, along with 9 µg transfection carrier DNA. The transfected cells were treated with UR241-2, starting at 20 µM (10-dose with 3-fold dilution) for 1 hour. The target engagement was then quantified by the NanoBRET assay. (E) Pie chart depicting the kinome profiling of UR241-2. The chart highlights kinases with >25% inhibition by UR241-2 at a concentration of 5 nM. Only 9 kinases out of 682 kinases were affected. STE = Homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases. TK = Tyrosine Kinase. TKL = Tyrosine Kinase-Like kinases. CK1 = Casein Kinase 1. AGC = PKA, PKG, and PKC families. CAMK = Calcium/calmodulin-dependent protein kinase. CMGC = CDK, MAPK, GSK3, and CLK families. (F) Cell viability assay of UR241-2 in THP-1 cells. Cells were treated with serial concentrations of UR241-2 (0.0013-20 μM) in the 96-well plate. After 48 hours, the cell viability was assessed using CellTiter-Glo 2.0 Cell Viability Assay. Vehicle = DMSO. (G) NF-κB reporter cell assay of UR241-2 in THP-1 NF-κB (Luc/GFP) reporter cells. Cells were pre-treated with serial concentrations of UR241-2 (0.032-4 μM) in the 96-well plate for 30 minutes and incubated with human IL-1β (10 ng/mL) for 6 hours. The activation levels of NF-κB were evaluated using ONE-Glo Luciferase Assay System. Vehicle = DMSO. IKKi = 20 μM IKK2 inhibitor (positive control). (H) Immunoblotting assay investigated IL-1 signaling in THP-1 cells treated with IRAK1/4 inhibitors. Cells were starved overnight in RPMI plus 0.5% FBS medium. Subsequently, cells were pre-treated with either vehicle (DMSO) or IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7; 4 μM) for 30 minutes and then incubated with human IL-1β (10 ng/mL) for 10 and 30 minutes. At each time point, cells were harvested and the phosphorylation and total levels of p65, p38, ERK, and Akt were analyzed. (I) CFU-C assays of MLL-AF9 cells treated with IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7). Live GFP + MLL-AF9 splenocytes were sorted and mixed with drugs (0.2 and 4 μM) in the methylcellulose-based medium. Colonies were counted after 7 days of culture. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (F) , (G) , and (I) . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.
    Irak4, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak4/product/MedChemExpress
    Average 93 stars, based on 3 article reviews
    irak4 - by Bioz Stars, 2026-02
    93/100 stars

    Images

    1) Product Images from "Targeting IL-1/IRAK1/4 signaling in Acute Myeloid Leukemia Stem Cells Following Treatment and Relapse"

    Article Title: Targeting IL-1/IRAK1/4 signaling in Acute Myeloid Leukemia Stem Cells Following Treatment and Relapse

    Journal: bioRxiv

    doi: 10.1101/2024.11.09.622796

    (A) Schematic of UR241-2 design and synthesis. This figure illustrates the logarithm of partition coefficient (LogP), topological polar surface area (tPSA), calculated logarithm of partition coefficient (CLogP), and logarithm of solubility (LogS). (B) Computer-simulated docking analyses for UR241-2 and PF06650833 against IRAK1 and IRAK4 using MedusaDock program. (C) The half maximal inhibitory concentration (IC 50 ) of UR241-2 for IRAK1 and IRAK4 measured by kinase profiling assay with protein suspension. UR241-2 underwent testing in 10-dose IC 50 duplicate mode, starting at 20 µM with 3-fold serial dilution. Reactions were carried out at 1 µM ATP. (D) The cellular IC 50 of UR241-2 for IRAK1 and IRAK4 measured by NanoBRET Target Engagement Assay in HEK293 cells. HEK293 cells were transiently transfected with 1 µg IRAK1 and IRAK4 NanoLuc Fusion Vectors, along with 9 µg transfection carrier DNA. The transfected cells were treated with UR241-2, starting at 20 µM (10-dose with 3-fold dilution) for 1 hour. The target engagement was then quantified by the NanoBRET assay. (E) Pie chart depicting the kinome profiling of UR241-2. The chart highlights kinases with >25% inhibition by UR241-2 at a concentration of 5 nM. Only 9 kinases out of 682 kinases were affected. STE = Homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases. TK = Tyrosine Kinase. TKL = Tyrosine Kinase-Like kinases. CK1 = Casein Kinase 1. AGC = PKA, PKG, and PKC families. CAMK = Calcium/calmodulin-dependent protein kinase. CMGC = CDK, MAPK, GSK3, and CLK families. (F) Cell viability assay of UR241-2 in THP-1 cells. Cells were treated with serial concentrations of UR241-2 (0.0013-20 μM) in the 96-well plate. After 48 hours, the cell viability was assessed using CellTiter-Glo 2.0 Cell Viability Assay. Vehicle = DMSO. (G) NF-κB reporter cell assay of UR241-2 in THP-1 NF-κB (Luc/GFP) reporter cells. Cells were pre-treated with serial concentrations of UR241-2 (0.032-4 μM) in the 96-well plate for 30 minutes and incubated with human IL-1β (10 ng/mL) for 6 hours. The activation levels of NF-κB were evaluated using ONE-Glo Luciferase Assay System. Vehicle = DMSO. IKKi = 20 μM IKK2 inhibitor (positive control). (H) Immunoblotting assay investigated IL-1 signaling in THP-1 cells treated with IRAK1/4 inhibitors. Cells were starved overnight in RPMI plus 0.5% FBS medium. Subsequently, cells were pre-treated with either vehicle (DMSO) or IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7; 4 μM) for 30 minutes and then incubated with human IL-1β (10 ng/mL) for 10 and 30 minutes. At each time point, cells were harvested and the phosphorylation and total levels of p65, p38, ERK, and Akt were analyzed. (I) CFU-C assays of MLL-AF9 cells treated with IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7). Live GFP + MLL-AF9 splenocytes were sorted and mixed with drugs (0.2 and 4 μM) in the methylcellulose-based medium. Colonies were counted after 7 days of culture. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (F) , (G) , and (I) . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.
    Figure Legend Snippet: (A) Schematic of UR241-2 design and synthesis. This figure illustrates the logarithm of partition coefficient (LogP), topological polar surface area (tPSA), calculated logarithm of partition coefficient (CLogP), and logarithm of solubility (LogS). (B) Computer-simulated docking analyses for UR241-2 and PF06650833 against IRAK1 and IRAK4 using MedusaDock program. (C) The half maximal inhibitory concentration (IC 50 ) of UR241-2 for IRAK1 and IRAK4 measured by kinase profiling assay with protein suspension. UR241-2 underwent testing in 10-dose IC 50 duplicate mode, starting at 20 µM with 3-fold serial dilution. Reactions were carried out at 1 µM ATP. (D) The cellular IC 50 of UR241-2 for IRAK1 and IRAK4 measured by NanoBRET Target Engagement Assay in HEK293 cells. HEK293 cells were transiently transfected with 1 µg IRAK1 and IRAK4 NanoLuc Fusion Vectors, along with 9 µg transfection carrier DNA. The transfected cells were treated with UR241-2, starting at 20 µM (10-dose with 3-fold dilution) for 1 hour. The target engagement was then quantified by the NanoBRET assay. (E) Pie chart depicting the kinome profiling of UR241-2. The chart highlights kinases with >25% inhibition by UR241-2 at a concentration of 5 nM. Only 9 kinases out of 682 kinases were affected. STE = Homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases. TK = Tyrosine Kinase. TKL = Tyrosine Kinase-Like kinases. CK1 = Casein Kinase 1. AGC = PKA, PKG, and PKC families. CAMK = Calcium/calmodulin-dependent protein kinase. CMGC = CDK, MAPK, GSK3, and CLK families. (F) Cell viability assay of UR241-2 in THP-1 cells. Cells were treated with serial concentrations of UR241-2 (0.0013-20 μM) in the 96-well plate. After 48 hours, the cell viability was assessed using CellTiter-Glo 2.0 Cell Viability Assay. Vehicle = DMSO. (G) NF-κB reporter cell assay of UR241-2 in THP-1 NF-κB (Luc/GFP) reporter cells. Cells were pre-treated with serial concentrations of UR241-2 (0.032-4 μM) in the 96-well plate for 30 minutes and incubated with human IL-1β (10 ng/mL) for 6 hours. The activation levels of NF-κB were evaluated using ONE-Glo Luciferase Assay System. Vehicle = DMSO. IKKi = 20 μM IKK2 inhibitor (positive control). (H) Immunoblotting assay investigated IL-1 signaling in THP-1 cells treated with IRAK1/4 inhibitors. Cells were starved overnight in RPMI plus 0.5% FBS medium. Subsequently, cells were pre-treated with either vehicle (DMSO) or IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7; 4 μM) for 30 minutes and then incubated with human IL-1β (10 ng/mL) for 10 and 30 minutes. At each time point, cells were harvested and the phosphorylation and total levels of p65, p38, ERK, and Akt were analyzed. (I) CFU-C assays of MLL-AF9 cells treated with IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7). Live GFP + MLL-AF9 splenocytes were sorted and mixed with drugs (0.2 and 4 μM) in the methylcellulose-based medium. Colonies were counted after 7 days of culture. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (F) , (G) , and (I) . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.

    Techniques Used: Solubility, Concentration Assay, Suspension, Serial Dilution, Transfection, Inhibition, Sterility, Viability Assay, Incubation, Activation Assay, Luciferase, Positive Control, Western Blot

    (A) CFU-C assays of paired primary human AML cells at diagnosis and in relapse (AML25). Cells were treated with IRAK1/4 inhibitors UR241-2, PF06650833, and IRAK4-IN-7 (0.2 and 4 μM) in methylcellulose-based medium. (B) CFU-C assays of paired diagnostic and relapsed AML34 cells, as described in (A). (C) CFU-C assays of primary human NBM CD34 + cells treated with UR241-2, PF06650833, and IRAK4-IN-7 (0.2 and 4 μM) in methylcellulose-based medium. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (A-C) . (D) Schematic of UR241-2 ex vivo treatment and transplantation using MLL-AF9 AML model. MLL-AF9 GFP + splenocytes were sorted and treated with vehicle (DMSO), UR241-2, and PF06650833 (4 μM) ex vivo for three days. These cells were transplanted into recipient C57BL/6J mice following treatment. Mice were sacrificed and assessed after four weeks. (E) Engraftment levels of MLL-AF9 GFP + cells in the bone marrow in recipients at four weeks after transplantation as described in (D) . Mean ± SD. Two-tailed Mann-Whitney test. (F) Spleen weights in recipients at four weeks after transplantation as described in (D) . Mean ± SD. Two-tailed Mann-Whitney test. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.
    Figure Legend Snippet: (A) CFU-C assays of paired primary human AML cells at diagnosis and in relapse (AML25). Cells were treated with IRAK1/4 inhibitors UR241-2, PF06650833, and IRAK4-IN-7 (0.2 and 4 μM) in methylcellulose-based medium. (B) CFU-C assays of paired diagnostic and relapsed AML34 cells, as described in (A). (C) CFU-C assays of primary human NBM CD34 + cells treated with UR241-2, PF06650833, and IRAK4-IN-7 (0.2 and 4 μM) in methylcellulose-based medium. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (A-C) . (D) Schematic of UR241-2 ex vivo treatment and transplantation using MLL-AF9 AML model. MLL-AF9 GFP + splenocytes were sorted and treated with vehicle (DMSO), UR241-2, and PF06650833 (4 μM) ex vivo for three days. These cells were transplanted into recipient C57BL/6J mice following treatment. Mice were sacrificed and assessed after four weeks. (E) Engraftment levels of MLL-AF9 GFP + cells in the bone marrow in recipients at four weeks after transplantation as described in (D) . Mean ± SD. Two-tailed Mann-Whitney test. (F) Spleen weights in recipients at four weeks after transplantation as described in (D) . Mean ± SD. Two-tailed Mann-Whitney test. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.

    Techniques Used: Diagnostic Assay, Ex Vivo, Transplantation Assay, Two Tailed Test, MANN-WHITNEY



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    irak4  (MedChemExpress)
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    MedChemExpress irak4
    (A) Schematic of UR241-2 design and synthesis. This figure illustrates the logarithm of partition coefficient (LogP), topological polar surface area (tPSA), calculated logarithm of partition coefficient (CLogP), and logarithm of solubility (LogS). (B) Computer-simulated docking analyses for UR241-2 and PF06650833 against IRAK1 and <t>IRAK4</t> using MedusaDock program. (C) The half maximal inhibitory concentration (IC 50 ) of UR241-2 for IRAK1 and IRAK4 measured by kinase profiling assay with protein suspension. UR241-2 underwent testing in 10-dose IC 50 duplicate mode, starting at 20 µM with 3-fold serial dilution. Reactions were carried out at 1 µM ATP. (D) The cellular IC 50 of UR241-2 for IRAK1 and IRAK4 measured by NanoBRET Target Engagement Assay in HEK293 cells. HEK293 cells were transiently transfected with 1 µg IRAK1 and IRAK4 NanoLuc Fusion Vectors, along with 9 µg transfection carrier DNA. The transfected cells were treated with UR241-2, starting at 20 µM (10-dose with 3-fold dilution) for 1 hour. The target engagement was then quantified by the NanoBRET assay. (E) Pie chart depicting the kinome profiling of UR241-2. The chart highlights kinases with >25% inhibition by UR241-2 at a concentration of 5 nM. Only 9 kinases out of 682 kinases were affected. STE = Homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases. TK = Tyrosine Kinase. TKL = Tyrosine Kinase-Like kinases. CK1 = Casein Kinase 1. AGC = PKA, PKG, and PKC families. CAMK = Calcium/calmodulin-dependent protein kinase. CMGC = CDK, MAPK, GSK3, and CLK families. (F) Cell viability assay of UR241-2 in THP-1 cells. Cells were treated with serial concentrations of UR241-2 (0.0013-20 μM) in the 96-well plate. After 48 hours, the cell viability was assessed using CellTiter-Glo 2.0 Cell Viability Assay. Vehicle = DMSO. (G) NF-κB reporter cell assay of UR241-2 in THP-1 NF-κB (Luc/GFP) reporter cells. Cells were pre-treated with serial concentrations of UR241-2 (0.032-4 μM) in the 96-well plate for 30 minutes and incubated with human IL-1β (10 ng/mL) for 6 hours. The activation levels of NF-κB were evaluated using ONE-Glo Luciferase Assay System. Vehicle = DMSO. IKKi = 20 μM IKK2 inhibitor (positive control). (H) Immunoblotting assay investigated IL-1 signaling in THP-1 cells treated with IRAK1/4 inhibitors. Cells were starved overnight in RPMI plus 0.5% FBS medium. Subsequently, cells were pre-treated with either vehicle (DMSO) or IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7; 4 μM) for 30 minutes and then incubated with human IL-1β (10 ng/mL) for 10 and 30 minutes. At each time point, cells were harvested and the phosphorylation and total levels of p65, p38, ERK, and Akt were analyzed. (I) CFU-C assays of MLL-AF9 cells treated with IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7). Live GFP + MLL-AF9 splenocytes were sorted and mixed with drugs (0.2 and 4 μM) in the methylcellulose-based medium. Colonies were counted after 7 days of culture. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (F) , (G) , and (I) . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.
    Irak4, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak4/product/MedChemExpress
    Average 93 stars, based on 1 article reviews
    irak4 - by Bioz Stars, 2026-02
    93/100 stars
    		  Buy from Supplier

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    (A) Schematic of UR241-2 design and synthesis. This figure illustrates the logarithm of partition coefficient (LogP), topological polar surface area (tPSA), calculated logarithm of partition coefficient (CLogP), and logarithm of solubility (LogS). (B) Computer-simulated docking analyses for UR241-2 and PF06650833 against IRAK1 and IRAK4 using MedusaDock program. (C) The half maximal inhibitory concentration (IC 50 ) of UR241-2 for IRAK1 and IRAK4 measured by kinase profiling assay with protein suspension. UR241-2 underwent testing in 10-dose IC 50 duplicate mode, starting at 20 µM with 3-fold serial dilution. Reactions were carried out at 1 µM ATP. (D) The cellular IC 50 of UR241-2 for IRAK1 and IRAK4 measured by NanoBRET Target Engagement Assay in HEK293 cells. HEK293 cells were transiently transfected with 1 µg IRAK1 and IRAK4 NanoLuc Fusion Vectors, along with 9 µg transfection carrier DNA. The transfected cells were treated with UR241-2, starting at 20 µM (10-dose with 3-fold dilution) for 1 hour. The target engagement was then quantified by the NanoBRET assay. (E) Pie chart depicting the kinome profiling of UR241-2. The chart highlights kinases with >25% inhibition by UR241-2 at a concentration of 5 nM. Only 9 kinases out of 682 kinases were affected. STE = Homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases. TK = Tyrosine Kinase. TKL = Tyrosine Kinase-Like kinases. CK1 = Casein Kinase 1. AGC = PKA, PKG, and PKC families. CAMK = Calcium/calmodulin-dependent protein kinase. CMGC = CDK, MAPK, GSK3, and CLK families. (F) Cell viability assay of UR241-2 in THP-1 cells. Cells were treated with serial concentrations of UR241-2 (0.0013-20 μM) in the 96-well plate. After 48 hours, the cell viability was assessed using CellTiter-Glo 2.0 Cell Viability Assay. Vehicle = DMSO. (G) NF-κB reporter cell assay of UR241-2 in THP-1 NF-κB (Luc/GFP) reporter cells. Cells were pre-treated with serial concentrations of UR241-2 (0.032-4 μM) in the 96-well plate for 30 minutes and incubated with human IL-1β (10 ng/mL) for 6 hours. The activation levels of NF-κB were evaluated using ONE-Glo Luciferase Assay System. Vehicle = DMSO. IKKi = 20 μM IKK2 inhibitor (positive control). (H) Immunoblotting assay investigated IL-1 signaling in THP-1 cells treated with IRAK1/4 inhibitors. Cells were starved overnight in RPMI plus 0.5% FBS medium. Subsequently, cells were pre-treated with either vehicle (DMSO) or IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7; 4 μM) for 30 minutes and then incubated with human IL-1β (10 ng/mL) for 10 and 30 minutes. At each time point, cells were harvested and the phosphorylation and total levels of p65, p38, ERK, and Akt were analyzed. (I) CFU-C assays of MLL-AF9 cells treated with IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7). Live GFP + MLL-AF9 splenocytes were sorted and mixed with drugs (0.2 and 4 μM) in the methylcellulose-based medium. Colonies were counted after 7 days of culture. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (F) , (G) , and (I) . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.

    Journal: bioRxiv

    Article Title: Targeting IL-1/IRAK1/4 signaling in Acute Myeloid Leukemia Stem Cells Following Treatment and Relapse

    doi: 10.1101/2024.11.09.622796

    Figure Lengend Snippet: (A) Schematic of UR241-2 design and synthesis. This figure illustrates the logarithm of partition coefficient (LogP), topological polar surface area (tPSA), calculated logarithm of partition coefficient (CLogP), and logarithm of solubility (LogS). (B) Computer-simulated docking analyses for UR241-2 and PF06650833 against IRAK1 and IRAK4 using MedusaDock program. (C) The half maximal inhibitory concentration (IC 50 ) of UR241-2 for IRAK1 and IRAK4 measured by kinase profiling assay with protein suspension. UR241-2 underwent testing in 10-dose IC 50 duplicate mode, starting at 20 µM with 3-fold serial dilution. Reactions were carried out at 1 µM ATP. (D) The cellular IC 50 of UR241-2 for IRAK1 and IRAK4 measured by NanoBRET Target Engagement Assay in HEK293 cells. HEK293 cells were transiently transfected with 1 µg IRAK1 and IRAK4 NanoLuc Fusion Vectors, along with 9 µg transfection carrier DNA. The transfected cells were treated with UR241-2, starting at 20 µM (10-dose with 3-fold dilution) for 1 hour. The target engagement was then quantified by the NanoBRET assay. (E) Pie chart depicting the kinome profiling of UR241-2. The chart highlights kinases with >25% inhibition by UR241-2 at a concentration of 5 nM. Only 9 kinases out of 682 kinases were affected. STE = Homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases. TK = Tyrosine Kinase. TKL = Tyrosine Kinase-Like kinases. CK1 = Casein Kinase 1. AGC = PKA, PKG, and PKC families. CAMK = Calcium/calmodulin-dependent protein kinase. CMGC = CDK, MAPK, GSK3, and CLK families. (F) Cell viability assay of UR241-2 in THP-1 cells. Cells were treated with serial concentrations of UR241-2 (0.0013-20 μM) in the 96-well plate. After 48 hours, the cell viability was assessed using CellTiter-Glo 2.0 Cell Viability Assay. Vehicle = DMSO. (G) NF-κB reporter cell assay of UR241-2 in THP-1 NF-κB (Luc/GFP) reporter cells. Cells were pre-treated with serial concentrations of UR241-2 (0.032-4 μM) in the 96-well plate for 30 minutes and incubated with human IL-1β (10 ng/mL) for 6 hours. The activation levels of NF-κB were evaluated using ONE-Glo Luciferase Assay System. Vehicle = DMSO. IKKi = 20 μM IKK2 inhibitor (positive control). (H) Immunoblotting assay investigated IL-1 signaling in THP-1 cells treated with IRAK1/4 inhibitors. Cells were starved overnight in RPMI plus 0.5% FBS medium. Subsequently, cells were pre-treated with either vehicle (DMSO) or IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7; 4 μM) for 30 minutes and then incubated with human IL-1β (10 ng/mL) for 10 and 30 minutes. At each time point, cells were harvested and the phosphorylation and total levels of p65, p38, ERK, and Akt were analyzed. (I) CFU-C assays of MLL-AF9 cells treated with IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7). Live GFP + MLL-AF9 splenocytes were sorted and mixed with drugs (0.2 and 4 μM) in the methylcellulose-based medium. Colonies were counted after 7 days of culture. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (F) , (G) , and (I) . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.

    Article Snippet: After serum-starvation, cells were pre-treated with UR241-2, PF06650833 (Selleck), and IRAK4-IN-7 (MCE) (4 µM) or vehicle (DMSO) for 30 minutes.

    Techniques: Solubility, Concentration Assay, Suspension, Serial Dilution, Transfection, Inhibition, Sterility, Viability Assay, Incubation, Activation Assay, Luciferase, Positive Control, Western Blot

    (A) CFU-C assays of paired primary human AML cells at diagnosis and in relapse (AML25). Cells were treated with IRAK1/4 inhibitors UR241-2, PF06650833, and IRAK4-IN-7 (0.2 and 4 μM) in methylcellulose-based medium. (B) CFU-C assays of paired diagnostic and relapsed AML34 cells, as described in (A). (C) CFU-C assays of primary human NBM CD34 + cells treated with UR241-2, PF06650833, and IRAK4-IN-7 (0.2 and 4 μM) in methylcellulose-based medium. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (A-C) . (D) Schematic of UR241-2 ex vivo treatment and transplantation using MLL-AF9 AML model. MLL-AF9 GFP + splenocytes were sorted and treated with vehicle (DMSO), UR241-2, and PF06650833 (4 μM) ex vivo for three days. These cells were transplanted into recipient C57BL/6J mice following treatment. Mice were sacrificed and assessed after four weeks. (E) Engraftment levels of MLL-AF9 GFP + cells in the bone marrow in recipients at four weeks after transplantation as described in (D) . Mean ± SD. Two-tailed Mann-Whitney test. (F) Spleen weights in recipients at four weeks after transplantation as described in (D) . Mean ± SD. Two-tailed Mann-Whitney test. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.

    Journal: bioRxiv

    Article Title: Targeting IL-1/IRAK1/4 signaling in Acute Myeloid Leukemia Stem Cells Following Treatment and Relapse

    doi: 10.1101/2024.11.09.622796

    Figure Lengend Snippet: (A) CFU-C assays of paired primary human AML cells at diagnosis and in relapse (AML25). Cells were treated with IRAK1/4 inhibitors UR241-2, PF06650833, and IRAK4-IN-7 (0.2 and 4 μM) in methylcellulose-based medium. (B) CFU-C assays of paired diagnostic and relapsed AML34 cells, as described in (A). (C) CFU-C assays of primary human NBM CD34 + cells treated with UR241-2, PF06650833, and IRAK4-IN-7 (0.2 and 4 μM) in methylcellulose-based medium. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (A-C) . (D) Schematic of UR241-2 ex vivo treatment and transplantation using MLL-AF9 AML model. MLL-AF9 GFP + splenocytes were sorted and treated with vehicle (DMSO), UR241-2, and PF06650833 (4 μM) ex vivo for three days. These cells were transplanted into recipient C57BL/6J mice following treatment. Mice were sacrificed and assessed after four weeks. (E) Engraftment levels of MLL-AF9 GFP + cells in the bone marrow in recipients at four weeks after transplantation as described in (D) . Mean ± SD. Two-tailed Mann-Whitney test. (F) Spleen weights in recipients at four weeks after transplantation as described in (D) . Mean ± SD. Two-tailed Mann-Whitney test. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.

    Article Snippet: After serum-starvation, cells were pre-treated with UR241-2, PF06650833 (Selleck), and IRAK4-IN-7 (MCE) (4 µM) or vehicle (DMSO) for 30 minutes.

    Techniques: Diagnostic Assay, Ex Vivo, Transplantation Assay, Two Tailed Test, MANN-WHITNEY