Figure 7.
MST1 negatively regulates IRAK1-dependent innate immune activation of NF-κB and proinflammatory cytokine production in MPN. (A) Interaction between IRAK1 and MST1 is assessed in HEK293T cell lysates by immunoblot analysis for MST1 and Flag-IRAK1 after Flag-IP. Input is shown below by immunoblot analysis for Flag-IRAK1, MST1, and β-actin as a loading control. (B) Gene expression measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in THP-1 cells 72 hours after transduction with control short hairpin RNA (shRNA; shCTRL, gray) or 2 independent STK4-targeting shRNAs (shSTK4_1/2, red/pink). Data are means ± SEM for 3 independent biological replicates. (C) Western blot showing the indicated protein and phospho-protein abundance in THP-1 cells 72 hours after transduction with shCTRL or shSTK4_1 vectors and stimulated with LPS (100 ng/mL) for the times indicated. Data are representative of 2 independent experiments. (D) Relative ubiquitination of TRAF6 in HEK293T cells transfected to express HA-ubiquitin (HA-Ub), Flag-TRAF6 and empty vector (−), MST1, or the kinase-dead mutant MST1-K59R, assessed by immunoblot analysis for HA-Ub after Flag-IP (HA-Ub-TRAF6). Input is shown by immunoblot for Flag, MST1, and β-actin as a loading control. Data are representative of 3 independent experiments. (E) Relative luciferase activity of a pNF-κB luciferase reporter construct (4× NF-κB–responsive elements upstream of minimal promoter and firefly luciferase) cotransfected in HEK293T cells with Flag-TRAF6 and either empty vector or MST1 expression vector. pRL-TK (Renilla luciferase reporter) is cotransfected as loading control, and firefly luciferase signal is normalized relative to Renilla. Data are means ± SEM for 3 independent biological replicates. Statistical significance was determined by 2-tailed Student t test. (F) Experimental schematic depicting strategy for generation of JAK2-V617F MPN model in 2 genotypes (Stk4+/+Stk3+/+ and Stk4+/−Stk3+/−) followed by IRAK1/4-inhibitor treatment, 4 mg/kg by intraperitoneal injection once every 3 days for 4 weeks. Wild-type controls, n = 12; V617F-Stk4+/+Stk3+/+, n = 7; and V617F-Stk4+/−Stk3+/−, n = 7. (G) GFP+ cell frequencies in peripheral blood measured by flow cytometry at 6 weeks after transplantation with JAK2-V617F–expressing cells of the indicated genotypes. (H) IL-1β abundance measured by ELISA in peripheral blood serum from mice of the indicated genotypes, before (untreated) or after (IRAK-inh) IRAK1/4-inhibitor treatment, as described in panel F. Statistical significance was determined by 1-way ANOVA followed by the post hoc Tukey test for multiple comparisons. (I) Western blot showing indicated protein and phospho-protein abundance in THP-1 cells 72 hours after transduction with shCTRL or shSTK4_1 vectors and stimulated with LPS (100 ng/mL) for 2 hours where indicated. Cells are pretreated with IRAK1/4-inhibitor (IRAK-inh) overnight at the indicated concentration, and this concentration was maintained during the LPS stimulation period. Data are representative of 2 independent experiments. *P < .05, **P < .01.

MST1 negatively regulates IRAK1-dependent innate immune activation of NF-κB and proinflammatory cytokine production in MPN. (A) Interaction between IRAK1 and MST1 is assessed in HEK293T cell lysates by immunoblot analysis for MST1 and Flag-IRAK1 after Flag-IP. Input is shown below by immunoblot analysis for Flag-IRAK1, MST1, and β-actin as a loading control. (B) Gene expression measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in THP-1 cells 72 hours after transduction with control short hairpin RNA (shRNA; shCTRL, gray) or 2 independent STK4-targeting shRNAs (shSTK4_1/2, red/pink). Data are means ± SEM for 3 independent biological replicates. (C) Western blot showing the indicated protein and phospho-protein abundance in THP-1 cells 72 hours after transduction with shCTRL or shSTK4_1 vectors and stimulated with LPS (100 ng/mL) for the times indicated. Data are representative of 2 independent experiments. (D) Relative ubiquitination of TRAF6 in HEK293T cells transfected to express HA-ubiquitin (HA-Ub), Flag-TRAF6 and empty vector (−), MST1, or the kinase-dead mutant MST1-K59R, assessed by immunoblot analysis for HA-Ub after Flag-IP (HA-Ub-TRAF6). Input is shown by immunoblot for Flag, MST1, and β-actin as a loading control. Data are representative of 3 independent experiments. (E) Relative luciferase activity of a pNF-κB luciferase reporter construct (4× NF-κB–responsive elements upstream of minimal promoter and firefly luciferase) cotransfected in HEK293T cells with Flag-TRAF6 and either empty vector or MST1 expression vector. pRL-TK (Renilla luciferase reporter) is cotransfected as loading control, and firefly luciferase signal is normalized relative to Renilla. Data are means ± SEM for 3 independent biological replicates. Statistical significance was determined by 2-tailed Student t test. (F) Experimental schematic depicting strategy for generation of JAK2-V617F MPN model in 2 genotypes (Stk4+/+Stk3+/+ and Stk4+/−Stk3+/−) followed by IRAK1/4-inhibitor treatment, 4 mg/kg by intraperitoneal injection once every 3 days for 4 weeks. Wild-type controls, n = 12; V617F-Stk4+/+Stk3+/+, n = 7; and V617F-Stk4+/−Stk3+/−, n = 7. (G) GFP+ cell frequencies in peripheral blood measured by flow cytometry at 6 weeks after transplantation with JAK2-V617F–expressing cells of the indicated genotypes. (H) IL-1β abundance measured by ELISA in peripheral blood serum from mice of the indicated genotypes, before (untreated) or after (IRAK-inh) IRAK1/4-inhibitor treatment, as described in panel F. Statistical significance was determined by 1-way ANOVA followed by the post hoc Tukey test for multiple comparisons. (I) Western blot showing indicated protein and phospho-protein abundance in THP-1 cells 72 hours after transduction with shCTRL or shSTK4_1 vectors and stimulated with LPS (100 ng/mL) for 2 hours where indicated. Cells are pretreated with IRAK1/4-inhibitor (IRAK-inh) overnight at the indicated concentration, and this concentration was maintained during the LPS stimulation period. Data are representative of 2 independent experiments. *P < .05, **P < .01.

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