Figure 4.
Proinflammatory cytokine TNF-α induces the expression of IL27Ra on HSCs via the ERK-ETS1 pathway. (A) Representative western blots showing the expression of IL27Ra, p-ERK, ERK, ETS1, and NF-κB p65 expression in young (2 months) and aged (28 months) hematopoietic stem and progenitor (c-Kit+) cells. (B) The histogram displays the expression of IL27Ra, Ets1, and NF-κB p65 in young HSCs (yHSC, 2 months) and aged HSCs (oHSC, 24 months). Data are shown as mean ± SEM. (C) Representative western blots showing the expression of ETS1, p-ERK, and ERK in young and aged HSCs. Freshly isolated 15 000 CD48− KSL cells from young (2 months) or aged (24 months) mice were lysed in SDS loading buffer. Western blot analysis was performed with indicated antibodies. (D) Representative western blots showing the expression of p-ERK and ETS1 in IL27Ra− or IL27Ra+ KSL cells from aged mice (26 months). Cells were sorted by flow cytometry and lysed in 2× SDS loading buffer. Lysis was completed by sonication and denatured by boiling. A total of 20 000 cells were used to detect p-ERK1/2 and ETS1. (E) The histogram displays the expression of IL27Ra expression in response to indicated cytokine stimulation (5 hours). Data are shown as mean ± SEM. (F) The schematic diagram (left) showing the experimental design to evaluate the influence of TNF-α on IL27Ra expression. The representative plots (right) showing the activation of IL27Ra in response to TNF-α stimulation. (G) Representative western blots showing the expression of IL27Ra and ETS1 in hematopoietic stem and progenitor cells in response to TNF-α ± ERKi ± IKKi treatment. c-Kit+ cells were either mock treated or treated with 50 ng/mL TNF-α for 24 hours in the presence or absence of 2 μM IKK inhibitor (IKKi, TPCA-1) or 10 μM ERK inhibitor (ERKi, PD98059). Western blot analysis was performed with indicated antibodies. (H) The schematic diagram (left) showing the experimental design to evaluate the influence of TNF-α ± ERKi on IL27Ra expression in vitro. The scatter plots (right) depict the percentage of IL27Ra+ cells upon TNF-α ± ERKi stimulation (50 ng/mL, 48 hours). Data are shown as mean ± SEM. (I-K) The schematic diagram showing the experimental design to evaluate the influence of TNF-α on IL27Ra expression in vivo. (I) Three 2-month-old WT mice were administered with TNF-α (5 μg/mouse, intraperitoneal injection) every 4 days. At the 14th day, mice were analyzed. (J) Representative western blots showing the expression of IL27Ra in response to TNF-α administration. (K) The percentage of IL27Ra+ HSCs in response to TNF-α administration. N = 3 mice per group. Data are shown as mean ± SEM. (L) The scatter plots depict the level of TNF-α in the bone marrow plasma from young (2 months) and old (24-28 months) mice. Data are shown as mean ± SEM.

Proinflammatory cytokine TNF-α induces the expression of IL27Ra on HSCs via the ERK-ETS1 pathway. (A) Representative western blots showing the expression of IL27Ra, p-ERK, ERK, ETS1, and NF-κB p65 expression in young (2 months) and aged (28 months) hematopoietic stem and progenitor (c-Kit+) cells. (B) The histogram displays the expression of IL27Ra, Ets1, and NF-κB p65 in young HSCs (yHSC, 2 months) and aged HSCs (oHSC, 24 months). Data are shown as mean ± SEM. (C) Representative western blots showing the expression of ETS1, p-ERK, and ERK in young and aged HSCs. Freshly isolated 15 000 CD48− KSL cells from young (2 months) or aged (24 months) mice were lysed in SDS loading buffer. Western blot analysis was performed with indicated antibodies. (D) Representative western blots showing the expression of p-ERK and ETS1 in IL27Ra− or IL27Ra+ KSL cells from aged mice (26 months). Cells were sorted by flow cytometry and lysed in 2× SDS loading buffer. Lysis was completed by sonication and denatured by boiling. A total of 20 000 cells were used to detect p-ERK1/2 and ETS1. (E) The histogram displays the expression of IL27Ra expression in response to indicated cytokine stimulation (5 hours). Data are shown as mean ± SEM. (F) The schematic diagram (left) showing the experimental design to evaluate the influence of TNF-α on IL27Ra expression. The representative plots (right) showing the activation of IL27Ra in response to TNF-α stimulation. (G) Representative western blots showing the expression of IL27Ra and ETS1 in hematopoietic stem and progenitor cells in response to TNF-α ± ERKi ± IKKi treatment. c-Kit+ cells were either mock treated or treated with 50 ng/mL TNF-α for 24 hours in the presence or absence of 2 μM IKK inhibitor (IKKi, TPCA-1) or 10 μM ERK inhibitor (ERKi, PD98059). Western blot analysis was performed with indicated antibodies. (H) The schematic diagram (left) showing the experimental design to evaluate the influence of TNF-α ± ERKi on IL27Ra expression in vitro. The scatter plots (right) depict the percentage of IL27Ra+ cells upon TNF-α ± ERKi stimulation (50 ng/mL, 48 hours). Data are shown as mean ± SEM. (I-K) The schematic diagram showing the experimental design to evaluate the influence of TNF-α on IL27Ra expression in vivo. (I) Three 2-month-old WT mice were administered with TNF-α (5 μg/mouse, intraperitoneal injection) every 4 days. At the 14th day, mice were analyzed. (J) Representative western blots showing the expression of IL27Ra in response to TNF-α administration. (K) The percentage of IL27Ra+ HSCs in response to TNF-α administration. N = 3 mice per group. Data are shown as mean ± SEM. (L) The scatter plots depict the level of TNF-α in the bone marrow plasma from young (2 months) and old (24-28 months) mice. Data are shown as mean ± SEM.

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