Journal: Journal of neuropathology and experimental neurology

Article Title: Effects of oncostatin M on human cerebral endothelial cells and expression in inflammatory brain lesions.

doi: 10.1093/jnen/60.11.1087

Figure Lengend Snippet: Fig. 2. HCEC transcribe mRNA for gp130, OSMR-b, LIFR, and IL-6R, but not for CNTFR. RT-PCR was performed on RNA extracted from HCEC and reverse-transcribed (RT1) using oligo(dT) primers. RNA samples not submitted to reverse transcription were used as negative controls (RT2). PCR results for gp130, LIFR, and OSMR-b are shown in the upper panel. Note that the expression level of LIFR was below that of gp130 and OSMR-b even when a 3-fold (33) higher amount of cDNA was used as starting material in the RT-PCR. PCR amplicons of CNTFR, IL-6R and b-actin (b-A), which was used as an internal control, are shown in the lower panel. RNA extracted from early postmortem human brain (hu Brain) served as a positive control for CNTFR. Identical results were obtained in all 5 HCEC preparations analyzed. Length of bands from DNA size marker (M, 1 kb DNA ladder, Gibco BRL) is indicated on the left (A). Surface detection of gp130 and OSMR-b, but not of LIFR, on HCEC. FACS analysis was performed as described in Materials and Methods to determine surface expression of gp130, OSMR-b, and LIFR on HCEC. Histograms show stain- ing with the respective receptor antibody (shaded) underlaid with the isotype-matched control (single line). Hela cells were

Article Snippet: For FACS analysis of adhesion molecules and OSM receptor subunits, HCEC were detached with 0.1% Trypsin/EDTA (Sigma), washed with PBS containing 0.1% BSA (Sigma) and 0.1% D ow nloaded from https://academ ic.oup.com /jnen/article/60/11/1087/2916229 by guest on 25 August 2024 J Neuropathol Exp Neurol, Vol 60, November, 2001 sodium azide, and incubated with primary mouse monoclonal antibodies against ICAM-1 (1:30; Bender MedSystems, Vienna, Austria), VCAM-1 (1:30; Biosource), gp130 (1:10), OSMRb (1:10, Santa Cruz Biotechnology, Santa Cruz, CA), and LIFR (1:10, Santa Cruz Biotechnology) for 30 min at 48C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Expressing, Control, Positive Control, Marker, Staining

Journal: The Journal of Biological Chemistry

Article Title: Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR

doi: 10.1016/j.jbc.2024.107251

Figure Lengend Snippet: Design of the cytokimera GIL-6 and GIO-6. A , schematic illustration of LIF within the trimeric LIF:gp130:LIFR complex, IL-6 in the hexameric 2xIL-6:2xIL-6R:2xgp130 complex, and OSM in the trimeric OSM:gp130:OSMR complex. IL-6 ( red ) was used as a scaffold for the generation of the cytokimera. Site 3 of IL-6 comprising of site 3-1, 3-2, and 3-3 was exchanged by site 3 of either LIF ( green ) leading to GIL-6 or by site 3 of OSM ( blue ) resulting in GIO-6. GIL-6 initially binds to D2/D3 of IL-6R via site 1. Subsequently, GIL-6 binds to D2/D3 of gp130 via site 2 of the IL-6 portion before GIL-6 binds to D3/D4 of the LIFR via site 3 of the LIF portion. GIL-6 forms a tetrameric GIL-6:IL-6R:gp130:LIFR complex. GIO-6 binds either to D2/D3 of the OSMR or to D3/D4 of the LIFR via site 3 of OSM, resulting in the tetrameric GIO-6:IL-6R:gp130:OSMR or GIO-6:IL-6R:gp130:LIFR complexes. B , superposed structure in ribbon and surface view of IL-6 ( red ) (1ALU) including the exchanged regions by LIF ( green ) or OSM ( blue ) for GIL-6 left and GIO-6 right , respectively. GIL-6 modelled in complex of the tetrameric GIL-6:IL-6R:gp130:LIFR complex (PDB 1ALU ; 2Q7N ; 1P9M ) in surface view.

Article Snippet: Anti-OSMR (catalog no. BAF4389), LIFR (catalog no. BAF249) (R&D Systems) were diluted 1:2000.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR

doi: 10.1016/j.jbc.2024.107251

Figure Lengend Snippet: GIL-6 induces JAK/STAT signaling and cellular proliferation via non-natural cytokine receptor complexes. A , proliferation of Ba/F3-gp130, Ba/F3-IL-6R:gp130, Ba/F3-gp130:OSMR, Ba/F3-gp130:LIFR, Ba/F3-IL-6R:gp130:OSMR, Ba/F3-IL-6R:gp130:OSMR, Ba/F3-IL-6R:gp130:LIFR cells without cytokine (−), with 100 ng/ml HIL-6, 10 ng/ml IL-6, 10 ng/ml LIF, 10 ng/ml OSM, 100 ng/ml GIL-6, 100 ng/ml GIO-6 and 100 ng/ml IC7. Data represents mean±S.D. of three independent experiments. For statistics the treated groups were compared with the untreated group by two-way ANOVA including Dunnet’s test for correction in multiple comparison. B , STAT3 activation in Ba/F3, Ba/F3-gp130, Ba/F3-IL-6R:gp130, Ba/F3-gp130:OSMR, Ba/F3-gp130:LIFR, Ba/F3-IL-6R:gp130:OSMR, Ba/F3-IL-6R:gp130:LIFR cells without cytokine (−) and after stimulation with 100 ng/ml HIL-6, 10 ng/ml IL-6, 10 ng/ml LIF, 10 ng/ml OSM, 100 ng/ml GIL-6, 100 ng/ml GIO-6 and 100 ng/ml IC7 for 20 min. C , STAT1, STAT3, STAT5, ERK, and Akt activation in Ba/F3-IL-6R:gp130:LIFR cells with the same conditions as for the STAT3 activation. D , STAT3 activation in heart, liver, and spleen after injection of 20 μg GIL-6 or GIO-6. 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. E , schematic illustration of IC7. Site 3 of IL-6 ( red ) comprising site 3-1, 3-2, and 3-3 was exchanged by site III of CNTF ( grey ) resulting in IC7. Consequently, IC7 forms tetrameric IC7:IL-6R:gp130:LIFR or IC7:IL-6R:gp130:OSMR complexes.

Article Snippet: Anti-OSMR (catalog no. BAF4389), LIFR (catalog no. BAF249) (R&D Systems) were diluted 1:2000.

Techniques: Comparison, Activation Assay, Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR

doi: 10.1016/j.jbc.2024.107251

Figure Lengend Snippet: Biological activity of cytokimeras GIL-6 and GIO-6 is comparable to natural cytokines. A , proliferation of Ba/F3-IL-6R:gp130, Ba/F3-gp130:LIFR, Ba/F3-gp130:OSMR, Ba/F3-IL-6R:gp130:LIFR, Ba/F3-IL-6R:gp130:OSMR cells in the presence and absence of increasing concentrations of GIL-6 (0.002–1000 ng/ml). The EC 50 values were calculated by fitting a non-linear regression curve. One representative experiment out of three is shown. B , proliferation of Ba/F3-IL-6R:gp130:LIFR cells in the presence and absence of increasing concentrations LIF (0.002–50 ng/ml), IL-6 (0.002–30 ng/ml) or IC7 (0.002–1000 ng/ml). One representative experiment out of three is shown. C , proliferation of Ba/F3-IL-6R:gp130, Ba/F3-gp130:LIFR, Ba/F3-gp130:OSMR, Ba/F3-IL-6R:gp130:LIFR, Ba/F3-IL-6R:gp130:OSMR cells in the presence and absence of increasing concentrations of GIO-6 (0.002–1000 ng/ml). One representative experiment out of three is shown. D , proliferation of Ba/F3-IL-6R:gp130:OSMR cells in the presence and absence of increasing concentrations of IC7 (0.002–2000 ng/ml) and proliferation of Ba/F3-gp130:OSMR cells in the presence of OSM (0.001–100 ng/ml). One representative experiment out of three is shown. E , STAT3 and ERK activation in Ba/F3-IL-6R:gp130:LIFR cells without cytokine (−) and after stimulation with increasing amounts of IL-6, LIF, GIL-6 or GIO-6 (0.2, 2, 20, 200 ng/ml) for 20 min. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT3, STAT3, phospho-ERK and ERK. Western blot data shows one representative experiment out of three. F , time-dependent STAT3 activation of Ba/F3-IL-6R:gp130:OSMR cells with OSM (10 ng/ml), IL-6 (10 ng/ml) or GIO-6 (100 ng/ml) and Ba/F3-IL-6R:gp130:LIFR cells with LIF (10 ng/ml), IL-6 (10 ng/ml) or GIL-6 (100 ng/ml) for the indicated time points. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3.

Article Snippet: Anti-OSMR (catalog no. BAF4389), LIFR (catalog no. BAF249) (R&D Systems) were diluted 1:2000.

Techniques: Activity Assay, Activation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR

doi: 10.1016/j.jbc.2024.107251

Figure Lengend Snippet: Cytokimera GIL-6 and GIO-6 are poor inducers of trans -signaling. A , proliferation of Ba/F3-gp130:LIFR cells in the presence and absence of fixed concentrations of sIL-6R (0 or 100 ng/ml) and increasing concentrations of IL-6 (0.002–1000 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-6R (0 or 100 ng/ml) and increasing concentrations of GIL-6 (0.002–2000 ng/ml). One representative experiment out of three is shown. C , proliferation of Ba/F3-gp130:LIFR cells in the presence and absence of fixed concentrations of sIL-6R (0 or 100 ng/ml) and increasing concentrations of GIO-6 (0.002–2000 ng/ml). One representative experiment out of three is shown. D , proliferation of Ba/F3-gp130:OSMR cells in the presence and absence of fixed concentrations of sIL-6R (0 or 100 ng/ml) and increasing concentrations of GIO-6 (0.002–2000 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 sIL-6R (0 or 100 ng/ml) and increasing concentrations of IC7 (0.002–1000 ng/ml). One representative experiment out of three is shown. F , STAT3 activation in Ba/F3-gp130:LIFR cells without cytokine (−), 10 ng/ml LIF, 100 ng/ml IL-6, 100 ng/ml GIL-6, 100 ng/ml GIO-6 and either with 100 ng/ml sIL-6R (+) or without (/). Equal amounts of protein (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. G , STAT3 activation in Ba/F3-gp130:OSMR cells without cytokine (−), 10 ng/ml OSM, 100 ng/ml IL-6, 100 ng/ml GIL-6, 100 ng/ml GIO-6 and either with 100 ng/ml sIL-6R (+) or without (/). Equal amounts of protein (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three.

Article Snippet: Anti-OSMR (catalog no. BAF4389), LIFR (catalog no. BAF249) (R&D Systems) were diluted 1:2000.

Techniques: Activation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR

doi: 10.1016/j.jbc.2024.107251

Figure Lengend Snippet: CNTF signals via the alternative CNTFR:gp130:OSMR complex but not via IL-6R:gp130:OSMR. A , proliferation of Ba/F3-CNTFR:gp130:LIFR or Ba/F3-CNTFR:gp130:OSMR cells with increasing concentrations of CNTF (0.0002–100 ng/ml). One representative experiment out of four is shown. B , proliferation of Ba/F3-gp130:LIFR or Ba/F3-gp130:OSMR cells in the presence of fixed concentrations of sIL-6R (100 ng/ml) and increasing concentrations of CNTF (0.05–100 ng/ml). One representative experiment out of three is shown. C and D , CNTF dose-dependent STAT3 activation of Ba/F3-CNTFR:gp130:LIFR ( C ), Ba/F3-CNTFR:gp130:OSMR ( D ) cells without cytokine (−) or in the presence of 10 ng/ml LIF or OSM, or with 0.1, 1, 10 or 100 ng/ml CNTF 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. E , proliferation of Ba/F3-gp130, Ba/F3-CNTFR:gp130:LIFR, Ba/F3-CNTFR:gp130:OSMR, Ba/F3-IL-6R:gp130:LIFR or Ba/F3-IL-6R:gp130:OSMR cells without cytokine (−) or in the presence of CNTF (0.5, 5 or 50 ng/ml), LIF (10 ng/ml), OSM (10 ng/ml) or IL-6 (10 ng/ml). Data represents mean ± S.D. of three independent experiments. For statistics, the treated groups were compared with the untreated group by two-way ANOVA including Dunnet’s test for correction in multiple comparisons. F , STAT3 activation Ba/F3-CNTFR:gp130:OSMR cells without cytokine (−), 10 ng/ml LIF, 10 ng/ml CNTF or 10 ng/ml OSM. 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. G and H , CNTF dose-dependent STAT3 activation of Ba/F3-IL-6R:gp130:LIFR ( G ) or Ba/F3-IL-6R:gp130:OSMR ( H ) cells without cytokine (−) or in the presence of 10 ng/ml LIF or OSM, or with 0.1, 1, 10 or 100 ng/ml CNTF 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. I , proliferation of Ba/F3-gp130:LIFR or Ba/F3-gp130:OSMR cells in the presence of increasing concentrations of Hyper-CNTF-IL-6R-Fc (0.02–8000 ng/ml). One representative experiment out of three is shown. J , schematic illustration of CNTF in tetrameric complexes consisting of CNTF:CNTFR:gp130:LIFR, CNTF:CNTFR:gp130:OSMR or CNTF:IL-6R:gp130:LIFR but not CNTF:IL-6R:gp130:OSMR.

Article Snippet: Anti-OSMR (catalog no. BAF4389), LIFR (catalog no. BAF249) (R&D Systems) were diluted 1:2000.

Techniques: Activation Assay, Western Blot