SEARCH

SEARCH BY CITATION

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_24655_sm_SuppFig1.tif1008KSupporting Figure 1: IL-6-induced α1-ACT mRNA expression is enhanced by glucocorticoids HepG2 cells were stimulated with IL-6 +/- dexamethasone for four hours. The expression of α1-ACT mRNA was analysed using qRT-PCR. IL-6-induced ACT mRNA was set to 100%. Data are given as mean of three independent experiments ± SD. Student's t-test for paired values: * p ≤ 0.05, ** p ≤ 0.01.
HEP_24655_sm_SuppFig2.tif2574KSupporting Figure 2: Densitometic analysis of figure 2C Levels of phosphorylated STAT3 were quantified using Multi Gauge software (Fujifilm Life Sciences, D'sseldorf, Germany). The histogram shows the ratio of phosphorylated STAT3 and total STAT3 protein per timepoint.
HEP_24655_sm_SuppFig3.tif1460KSupporting Figure 3: Glucocorticoids don't alter nuclear translocation of STAT3 HepG2 cells were stimulated with IL-6 +/- dexamethasone for the indicated times. Nuclear and cytoplasmic extracts were prepared and total and phosphorylated STAT3 were analysed by Western blotting. Staining for GAPDH and Lamin show the specificity and purity the fractions. Cell fractionation All fractionation and centrifugation steps were performed at 4 °C. HepG2 cells grown in ten cm plates, were harvested in 1 ml PBS and centrifuged for five minutes (500 rpm). Cell pellets were resuspended in 100 μl hypotonic lysisbuffer (10 mM Tris/HCL, pH 7.5, 10 mM NaCl, 3 mM MgCl2 supplemented with 10 μg/ml of each aprotinin, leupeptin and pepstatin as well as 0.8 μM Pefabloc, 1 mM Na3VO4, 1 mM EDTA and 1 mM DTT) and incubated for 15 minutes. Cells were centrifuged for ten minutes (2000 rpm). The supernatant containing the cytoplasmic fraction was centrifuged again for 10 minutes (2000 rpm) to clear the lysate. Pellets from former step were resolved in 500 μl nuclear isolation buffer (0.5 % NP-40, 10 mM Tris/HCL, pH 7.5, 10 mM NaCl, 3 mM MgCl2 supplemented with with 10 μg/ml of each aprotinin, leupeptin and pepstatin as well as 1 mM Na3VO4, 1 mM EDTA, 1 mM DTT) and incubated for five minutes. Cells were centrifuged twice for five minutes (2000 rpm) and the nuclear pellet was resuspended in 50 μl nuclear lysis buffer (2 % Triton X-100, 20 mM Tris/HCl, pH 7.5, 280 mM NaCl supplemented with 10 μg/ml of each aprotinin, leupeptin and pepstatin as well as 1 mM Na3VO4, 10 mM NaF, 1 mM EDTA, 1 mM DTT). After incubation for 30 minutes the cells were centrifuged for ten minutes (13000 rpm) to clear the lysate.
HEP_24655_sm_SuppFig4.tif1206KSupporting Figure 4: Glucocorticoids don't affect SOCS3 protein stability In order to study the modulation of SOCS3 protein stability in more detail an expression system based on a modified pTET splice vector system described by Xu et al. (Nucleic Acids Res. 1998, 26: 558-565) was established. pTET-HA-SOCS3 codes for the full length human SOCS3 mRNA under the control of a tetracycline sensitive (Tet-off) promoter. Using this experimental approach can directly monitor the decay of SOCS3 as described for murine SOCS3 by Ehlting et al. (J Immunol. 2007, 178: 2813-2826). HepG2 cells were co-transfected with 1.0 μg of the pTET-HA-SOCS3 expression vector and 1.5 μg of the pTET-off vector. 24 hours after transfection cells were treated with doxycycline (4 μg/ml) as indicated. Cells were lysed and 60 μg of protein was analyzed by Western blotting using antibodies specific for SOCS3 and GAPDH. Control: lysates from untransfected cells.
HEP_24655_sm_SuppFig5.tif1134KSupporting Figure 5: Murine embryonic fibroblasts respond to dexamethasone and IL-6 and can be used as a cellular model system to analyse SOCS3-dependency A) Glucocorticoids reduce IL-6-induced SOCS3 protein in MEF cells. MEF cells stimulated with IL-6 and soluble IL-6R +/- dexamethasone for the indicated times. SOCS3 and HSP70 protein expression were analysed by Western blotting. B) MEF WT and MEF SOCS3-/- cells stimulated with IL-6 and soluble IL-6R for 60 min. SOCS3 was analysed by Western blotting.
HEP_24655_sm_SuppFig6.tif2557KSupporting Figure 6: Glucocorticoids don't induce SAA gene expression in vivo A) RNA was isolated from whole mouse liver extracts after the indicated treatment. The expression of SAA mRNA was analysed via qRT-PCR. Data are given as mean of n = 3 animals per group ± SD. B) Serum SAA concentration was measured by ELISA. Data are given as mean of n = 3 animals per group ± SD.
HEP_24655_sm_SuppFig7.tif2321KSupporting Figure 7: The enhancing effect of glucocorticoids on the IL-6-induced α2M-promoter activation depends on the SOCS -recruiting motif within gp130 HepG2 cells were transfected with a α2-macroglobulin reporter construct and a galactosidase expression vector. Five hours after transfection cells were starved. Stimulation occurred via chimeric Epo-receptor constructs in A) EpoR/gp130(YYYYYY) and in B) EpoR/gp130(YFYYYY) with Epo (7 U/ml) either in the absence or presence of dexamethasone. Maximal induction of the α2-macroglobulin promoter in cells expressing EpoR/gp130(YYYYYY) was set to 100 %. Data are given as means of three independent experiments ± SD. Student's t-test for paired values: ** p ≤ 0.01, *** p ≤ 0.001.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.