Fig 2: Biological activity of GIL-11 is comparable to IL-11 and LIF.a Proliferation of Ba/F3, Ba/F3-gp130, Ba/F3-gp130:IL-11R, Ba/F3-gp130:LIFR, Ba/F3-gp130:OSMR, Ba/F3-gp130:IL-11R:OSMR, Ba/F3-gp130:IL-6R:LIFR, Ba/F3-gp130:LIFR:IL-11R cells in the presence and absence of increasing concentrations of GIL-11 (0.0005–500 ng/ml). b Proliferation of Ba/F3-gp130:IL-11R:LIFR cells in the presence and absence of increasing concentrations of LIF (0.000025–200 ng/ml). c Proliferation of Ba/F3-gp130:IL-11R:LIFR cells in the presence and absence of increasing concentrations of IL-11 (0.000025–500 ng/ml). a–c Error bars define ±SEM. One representative experiment with three biological replicates out of three is shown. d STAT3 and ERK activation in Ba/F3-gp130:LIFR:IL-11R cells without cytokine (-) and after stimulation with increasing amounts of GIL-11 (1, 10, 100, 1000 ng/ml), LIF (0.1, 1, 10, 100 ng/ml) and IL-11 (1, 10, 100, 1000 ng/ml) for 15 min. Equal amounts of proteins (50 µg/lane) were analyzed via specific antibodies detecting phospho-STAT3, STAT3, ERK and phospho-ERK. Western blot data shows one representative experiment out of three. e STAT1,3,5,6 activation in Ba/F3-gp130:IL-11R:LIFR cells without cytokine (-) and after stimulation with 50 ng/ml HIL-11, 50 ng/ml IL-11, 10 ng/ml LIF, 10 ng/ml OSM, 500 ng/ml GIL-11 for 15 min. Equal amounts of proteins (50 µg/lane) were analyzed via specific antibodies detecting phospho-STATs and STATs. Western blot data shows one representative experiment out of three. f Time-dependent STAT3 activation of Ba/F3-gp130:IL-11R:LIFR cells with the cytokines IL-11 (200 ng/ml), LIF (20 ng/ml) and GIL-11 (200 ng/ml) for the indicated time. Equal amounts of proteins (50 µg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3.

Fig 3: GIL-11 induce JAK/STAT signaling and cellular proliferation via the non-natural gp130:IL-11R:LIFR cytokine receptor complex.a Proliferation of Ba/F3-gp130, Ba/F3-gp130-IL-11R, Ba/F3-gp130:LIFR, Ba/F3-gp130:OSMR, Ba/F3-gp130:IL-11R:OSMR, Ba/F3-gp130:IL-6R:LIFR, Ba/F3-gp130:LIFR:IL-11R cells without cytokine (-), with 50 ng/ml HIL-11, 50 ng/ml IL-11, 50 ng/ml IL-6, 10 ng/ml LIF, 10 ng/ml OSM, 500 ng/ml GIL-11. Data represent ±SEM of three independent experiment out of three with three technical replicates each. Two-way ANOVA, including Dunnet’s correction was used for statistics. b STAT3 activation in Ba/F3, Ba/F3-gp130, Ba/F3-gp130_IL-11R, Ba/F3-gp130:LIFR, Ba/F3-gp130:OSMR, Ba/F3-gp130:IL-11R:OSMR, Ba/F3-gp130:IL-6R:LIFR, Ba/F3-gp130:LIFR:IL-11R cells without cytokine (-) and after stimulation with 50 ng/ml HIL-11, 50 ng/ml IL-11, 10 ng/ml LIF, 10 ng/ml OSM, 500 ng/ml GIL-11 for 15 min. Equal amounts of proteins (50 µg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three.

Fig 4: GIL-11:sIL-11 complexes induce trans-signaling via gp130:LIFR.a Proliferation of Ba/F3-gp130:LIFR cells in the presence and absence of fixed concentrations of sIL-11R (0 or 100 ng/ml) and increasing concentrations of IL-11 (1–2000 ng/ml). One representative experiment out of three is shown. b Proliferation of Ba/F3-gp130:LIFR cells in the presence and absence of fixed concentrations of sIL-11R (0 or 100 ng/ml) and increasing concentrations of GIL-11 (1–2000 ng/ml). One representative experiment out of three is shown. c STAT3 activation in Ba/F3-gp130:LIFR cells without cytokine (-) and after stimulation with HIL-11 (50 ng/ml), GIL-11 (1 µg/ml), GIL-11 (1 µg/ml):sIL-11R (2 µg/ml), GIL-11 (1 µg/ml):sIL-11R (2 µg/ml):sgp130Fc (10 µg/ml) for 15 min. Equal amounts of proteins (50 µg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. d Proliferation of Ba/F3-gp130:LIFR cells in the presence and absence of fixed concentrations of IL-11 (0.5 µg/ml):sIL-11R (1 µg/ml) or GIL-11 (0.5 µg/ml):sIL-11R (1 µg/ml) and increasing concentrations of sgp130Fc (1–10,000 ng/ml). One representative experiment out of three is shown. e Proliferation of Ba/F3-gp130:LIFR cells in the presence and absence of fixed concentrations of LIF (10 ng/ml) and increasing concentrations of GIL-11 (1–2000 ng/ml) (green). Proliferation of Ba/F3-gp130:IL-11R cells in the presence and absence of fixed concentrations of IL-11 (100 ng/ml) and increasing concentrations of GIL-11 (1–2000 ng/ml) (red). Error bars define ±SEM. One representative experiment with three biological replicates out of three is shown.

Fig 5: Human GIL-11 transmits signal transduction via the non-natural murine gp130:IL-11R:LIFR cytokine receptor complex.a STAT3 activation in un-transfected and transfected with a cDNA coding for murine IL-11R murine myoblasts (C2C12) without cytokine (-) and after stimulation with HIL-11 (200 ng/ml), IL-11 (200 ng/ml), LIF (10 ng/ml), GIL-11 (200 ng/ml) for 15 min. Equal amounts of proteins (50 µg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. b STAT3 activation in murine fibroblast NIH/3T3 without cytokine (-) and after stimulation with increasing amounts of GIL-11 (1, 10, 100, 1000 ng/ml), LIF (0.1, 1, 10, 100 ng/ml) and IL-11 (1, 10, 100, 1000 ng/ml) for 20 min. Equal amounts of proteins (50 µg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. c STAT3 activation in heart, liver and spleen after injection of 5, 10 or 20 µg/ml GIL-11. Mice were sacrificed 30 min after intraperitoneal cytokine injection. Equal amounts of proteins (50 µg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three.