Figure 1
Defective responses of XIAP-deficient macrophages to dectin-1 agonist but not to LPS Pam3CSK4, poly (I:C), or R848. (A-C) Comparable LPS-, Pam3CSK4-, R848-, or poly (I:C)-induced TNF-α and IL-6 production in control and Xiap−/− cells. Control and Xiap−/− BMDMs were activated with LPS (250 ng/mL), Pam3CSK4 (500 ng/mL), R848 (2 μg/mL), or poly (I:C) (50 μg/mL), and the secretion of (A,C) TNF-α and (B) IL-6 was quantitated 18 hours later by enzyme-linked immunosorbent assay (ELISA) using IL-6 DuoSet and TNF-α DuoSet. (D-F) XIAP-knockout reduced dectin-1–induced cytokine secretion. Control and Xiap−/− BMDMs were seeded overnight and treated with zymosan depleted (100 μg/mL) to activate dectin-1. The (D) TNF-α, (E) IL-6, and (F) IL-10 generated were quantitated 16 hours after activation by ELISA. (G) Knockdown of XIAP in human macrophages. Human macrophages were differentiated from peripheral monocytes and were transfected with control and XIAP-siRNA. The expression levels of XIAP were determined 48 hours after transfection. (H-I) Reduced dectin-1–induced TNF-α and IL-6 in XIAP-knockdown human macrophages. Control and XIAP-knockdown human macrophages (48 hours after transfection) were seeded overnight and treated with zymosan depleted (100 μg/mL) to activate dectin-1. The TNF-α and IL-6 generated were quantitated 16 hours after activation by ELISA. **P < .01 and ***P < .001 for paired Student t test. Each data point represents mean of triplicate determinations in a single experiment, and each experiment (A-I) was repeated 3 times with similar results. (J-L) Impaired NF-κB activation and p38 MAPK phosphorylation in Xiap−/− BMDMs stimulated with dectin-1 ligands. (J,L) Total cell lysates and (K) nuclear extracts were prepared from BMDMs stimulated with (J-K) zymosan depleted or (L) curdlan at the indicated time points. (J,L) IκBα and p38 phosphorylation in total cell lysates and (K) the entry of p65 in nuclear extracts were determined by immunoblotting. HSP90 and HDAC2 were the internal controls for the total cell lysate or nuclear extract, respectively. (J-L) were repeated twice with similar results.

Defective responses of XIAP-deficient macrophages to dectin-1 agonist but not to LPS Pam3CSK4, poly (I:C), or R848. (A-C) Comparable LPS-, Pam3CSK4-, R848-, or poly (I:C)-induced TNF-α and IL-6 production in control and Xiap−/− cells. Control and Xiap−/− BMDMs were activated with LPS (250 ng/mL), Pam3CSK4 (500 ng/mL), R848 (2 μg/mL), or poly (I:C) (50 μg/mL), and the secretion of (A,C) TNF-α and (B) IL-6 was quantitated 18 hours later by enzyme-linked immunosorbent assay (ELISA) using IL-6 DuoSet and TNF-α DuoSet. (D-F) XIAP-knockout reduced dectin-1–induced cytokine secretion. Control and Xiap−/− BMDMs were seeded overnight and treated with zymosan depleted (100 μg/mL) to activate dectin-1. The (D) TNF-α, (E) IL-6, and (F) IL-10 generated were quantitated 16 hours after activation by ELISA. (G) Knockdown of XIAP in human macrophages. Human macrophages were differentiated from peripheral monocytes and were transfected with control and XIAP-siRNA. The expression levels of XIAP were determined 48 hours after transfection. (H-I) Reduced dectin-1–induced TNF-α and IL-6 in XIAP-knockdown human macrophages. Control and XIAP-knockdown human macrophages (48 hours after transfection) were seeded overnight and treated with zymosan depleted (100 μg/mL) to activate dectin-1. The TNF-α and IL-6 generated were quantitated 16 hours after activation by ELISA. **P < .01 and ***P < .001 for paired Student t test. Each data point represents mean of triplicate determinations in a single experiment, and each experiment (A-I) was repeated 3 times with similar results. (J-L) Impaired NF-κB activation and p38 MAPK phosphorylation in Xiap−/− BMDMs stimulated with dectin-1 ligands. (J,L) Total cell lysates and (K) nuclear extracts were prepared from BMDMs stimulated with (J-K) zymosan depleted or (L) curdlan at the indicated time points. (J,L) IκBα and p38 phosphorylation in total cell lysates and (K) the entry of p65 in nuclear extracts were determined by immunoblotting. HSP90 and HDAC2 were the internal controls for the total cell lysate or nuclear extract, respectively. (J-L) were repeated twice with similar results.

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