Serum concentration dependent proliferation of CYLD+/+ and CYLD−/− primary MEFs.

<p>(<b>A–D</b>). Measurement of the growth rate of MEFs by cell counting after serum withdrawal (24 hours) followed by re-addition of FCS: 0, 1, 5 and 10% over a period of 24–96 hours. (<b>E</b>). Analysis of the levels of cyclin D1 and tubulin in primary CYLD+/+ compared to CYLD −/− MEFs in the absence (0%) or presence of 10% FCS for 48 hours. (<b>F</b>). Confocal plane of Bcl-3 (red) and DAPI (blue) in CYLD+/+ (upper) and CYLD−/− (lower) MEFs after serum withdrawal (24 hours) followed by re-addition of 10% FCS for 24 hours. (<b>G–J</b>). Measurement of the growth rate of MEFs by cell counting treated with serum free for 24 hours before re-addition of 1% FCS together with EGF (100 ng/ml), LPA (1 µM), TPA (100 nM) and or TNF-α (100 ng/ml) over a period of 24–96 hours.</p

CYLD gene expression is regulated at the transcriptional level by serum.

<p>(<b>A</b>). Analysis of the levels of CYLD, cyclin D1 and tubulin in primary CYLD+/+ MEFs in 10% FCS (control) or serum deprived cells over a period of 24–72 hours. (<b>B–C</b>). Analysis of the levels of CYLD and tubulin in primary CYLD+/+ MEFs in 10% FCS (control) or serum deprived cells for 24 hours (0%) or re-addition of FCS (10%) to the cells over a period of 24–72 hours. (<b>D</b>). Analysis of the levels of CYLD and tubulin in primary CYLD+/+ MEFs in 10% FCS over a period of 0.5–24 hours. (<b>E</b>). CYLD gene expression by using qRT-PCR upon withdrawal (0% FCS) or re-addition of serum to the cell cultures for 1 or 4 hours. (<b>F</b>). Analysis of the levels of CYLD and tubulin in primary CYLD+/+ MEFs after serum deprivation 24 hours before re-addition of 10% FCS (control) or 10% FCS together with 0.5–1.0 µg/ml actinomycin D for 12 hours.</p

Effects of p38MAPK inhibition to serum mediated CYLD expression.

<p>(<b>A–C</b>). Western blot analysis of CYLD and tubulin expression in serum starved WT MEFs (24 hours) and readdition of 10% FCS for 24 hours in the absence or presence of SB-203580 (500 nM), PD 98058 (25 µM), UO126 (20 µM), SP600125 (20 µM) or solvent (DMSO). (<b>D</b>). Western blot analysis of active and total p38MAPK in WT MEFs in the absence (24 hours) or readdition of 10% FCS for 30, 60 or 120 minutes. (<b>E</b>). Lysates from CYLD+/+ MEFs were examined by ChIP assay in the absence or presence of solvent (DMSO) or SB203580 (500 nM for 1 hour). Cells were serum starved for 24 hours and one hour before readdition of serum, SB203580 (500 nM for 1 hour) or solvent (DMSO) was added to the cell culture. Cell lysates were chromatin immunoprecipitated using anti-SRF antibody and a PCR primer pair corresponding to the promoter of the CYLD gene was used. Immunoprecipitation (ChIP) using specific antibodies; IgG: IP using negative control rabbit immunoglobulin; Input: 10% of the cell lysate used for the IP is shown. (F). Lysates from CYLD+/+ MEFs were examined by ChIP assay using anti-pSRF or anti- pELK1 and a PCR primer pair corresponding to the promoter of the CYLD gene (270 bp). Immunoprecipitation (ChIP) using specific antibodies; IgG: IP using negative control rabbit immunoglobulin; Input: 10% of the cell lysate used for the IP is shown. (<b>G</b>). Cell counting of CYLD+/+ MEFs in serum starved WT MEFs (24 hours) before re-addition of 10% FCS for 48 hours in the absence or presence of solvent (DMSO) or SB203580 (500 nM for 1 hour).</p

Recruitment of SRF to the promoter of CYLD induced by serum.

<p>(<b>A</b>). Location of two serum response elements identified at CYLD promoter. (<b>B</b>). Lysates from CYLD+/+ MEFs were examined by ChIP assay using an anti- SRF (H-300, Santa Cruz) and a PCR primer pair corresponding to the promoter of the CYLD gene (270 bp). Immunoprecipitation (ChIP) using specific antibodies; IgG: IP using negative control rabbit immunoglobulin; Input: 10% of the cell lysate used for the IP is shown. (<b>C</b>). Reporter assays revealing inducible CYLD promoter (-1297 to -1) activity in MEF cells; (p1194SRE), whereas mutation of the consensus SRF binding site (p1194ΔSRE) led to reduced promoter activity. (<b>D</b>). Western blot analysis of SRF and tubulin expression in the presence of 10% FCS for 48 hours (control) or cells incubated in the absence of serum over a period of 24–72 hours (0% FBS). (<b>E</b>). Western blot analysis of SRF, CYLD and tubulin expression in scrambled siRNA control transfected cells or cells transfected with the SRF siRNA nucleotides. (<b>F</b>). Western blot analysis of SRF, CYLD and tubulin expression in cells transiently transfected with the SRF siRNA nucleotides after serum withdrawal (24 hours) and re-addition of FCS over a period of 24–72 hours. Control cells are transiently transfected with the scramble siRNA nucleotides after re-addition of FCS for 48 hours. (<b>G</b>). Cell counting of CYLD+/+ MEFs transiently transfected with scramble or SRF siRNA nucleotides in the presence of 10% FCS over period of 24–48 hours.</p

Effects of TNF-α mediated apoptosis in CYLD +/+ and CYLD−/− MEFs.

<p>(<b>A</b>). The apoptotic cells were analyzed by detecting the gradual degradation of internucleosomal DNA with the DNA binding fluorescent dye propidium iodide in WT and CYLD-KO MEF cells in the absence or presence of 10% FCS over a period of 24–96 hours. (<b>B</b>). The apoptotic cells were analyzed by detecting the gradual degradation of internucleosomal DNA with the DNA binding fluorescent dye propidium iodide in WT and CYLD-KO MEF cells treated with TNF-α (100 ng/ml) for 12 hours, cyclohexamide (10 µg/ml) for 12 hours or a combination of TNF-α and cyclohexamide (100 ng/ml respective 10 µg/ml) for 12 hours.</p

Collagen I dependent 2D and 3D cell migration is regulated by CD47.

<p>(A) Wound healing assay; Int cells were pre-incubated or not with 20 µg/ml of the CD47 functional blocking antibody B6H12 for 2 hours, IgG or β2 integrin antibody for 20 minutes, or 100 µM of the COX-2 specific inhibitor NS-398 for 30 minutes, thereafter cells were plated onto 10 µg/ml collagen I coated dishes for 2 hours, after which a wound was made in the monolayer, and the cells were allowed to migrate for 18 hours. Pictures of the wound were taken after 0 and 18 hours. The wound closure was measured and is presented as the percentage of wound closure as compared to time zero. (B, C) 3D cell migration assay; (B) Cells were pre-incubated or not with 20 µg/ml of the CD47 functional blocking antibody B6H12 for 2 hours, IgG or the β2 integrin antibody for 20 minutes, or 100 µM of the COX-2 specific inhibitor NS-398 for 30 minutes after which 250,000 cells from each group were allowed to migrate through a 3 mg/ml collagen I gel and across an 8.0 µm micropore membrane for 18 hours. (C) Int 407 cells were transfected with 50 nM siRNA against CD47 (as indicated) or with scrambled control siRNA, for 48 hours. Thereafter 250,000 cells from each group were allowed to migrate through a 3 mg/ml collagen I gel and across an 8.0 µm micropore membrane for 18 hours. Cell migration was examined by staining with crystal violet blue and measuring the absorbance at 590 nm. The data are given as percent of control and represent means ± SE of five separate experiments. The statistical analyses were performed with unpaired Students t-test; *P<0.05, **P<0.01 relative to the control.</p

Collagen I induces COX-2 expression through a PTX dependent G-protein.

<p>(A) Int 407 cells were incubated with or without 500 ng/ml PTX for 2 hours, after which they were plated out onto 10 µg/ml collagen I or 6% BSA (control) coated dishes for 1 hour. Adherent cells were then lysed and analysed for COX-2 expression by Western blotting as previously described. (B) Cells were transiently transfected with empty vector or vectors expressing small inhibitor peptides against either Gα_i1-2 or Gα_i3 before plated onto 10 µg/ml collagen I coated dishes for 1 hour. Adherent cells were the lysed and analysed for COX-2 expression by Western blotting. All membranes were re-probed for actin to ensure equal loading. The accumulated data of the densitometric analyses are given as percent of control and represent means ± SE of four separate experiments. The statistical analyses were performed with unpaired Students t-test; *P<0.05, **P<0.01 relative to the control and compared to collagen I treatment.</p

CYLD diminishes NF-κB-dependent IL-6 production, ROS production and pathogen control of Lm-infected macrophages.

<p>(A–C) BMDM were isolate from WT and Cyld^−/− mice and stimulated with IFN-γ (100 U/ml). Indicated groups were infected with Lm (MOI of 5∶1) and treated with IKK inhibitor (10 µM for 4 h followed by 1 µM for 20 h), respectively. (A) After 24 h, the amount of intracellular Lm was determined in 1×10⁶ BMDM. (B) ROS production was analysed by flow cytometry in Lm-infected macrophages 24 h after infection. (C) The supernatant was harvested from uninfected and infected macrophages after 24 h and analysed for IL-6 by CBA. In (A–C), data show the mean ± SD of triplicate wells; * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001. (D) Proteins were isolated from uninfected and Lm-infected BMDM at the indicated time points. Cells were stimulated with IFN-γ and IKK inhibitor VII as indicated. WBs were incubated with α-p-p65, α-p65, and α-GAPDH as loading control. Representative WBs from a total of three independent experiments are shown. (E) Quantification of p-p65 intensity (± SD) was performed from WB data of uninfected and Lm-infected BMDM, which were stimulated as described in (D). The results present pooled data from 3 independent experiments.</p

IL-6 neutralization abolishes increased STAT3 activation, fibrin production, survival and pathogen control in Lm-infected Cyld^−/− mice.

<p>(A) The survival rates of rat IgG and α-IL-6-treated Lm-infected WT and Cyld^−/− mice (n = 7 per experimental group) are shown. The survival rate of IgG-treated Cyld^−/− mice (p<0.05) but not of the other groups was significantly increased as compared to rat IgG-treated WT mice. (B) CFUs were determined in the liver of Lm-infected rat IgG and IL-6-treated WT and Cyld^−/− mice at day 5 p.i. (n = 5 per experimental group; * p<0.05). Data show the mean ± SD from one of two representative experiments. (C) Proteins were isolated from livers of infected rat IgG and IL-6-treated WT and Cyld^−/− mice (n = 3 per experimental group) at day 5 p.i. WB analysis for CYLD, pSTAT3, fibrin and GAPDH was performed and representative data are shown. (D, E). Quantification of hepatic pSTAT3 (D) and fibrin (E) (± SD) was performed from WB data of rat IgG and α-IL-6-treated WT and Cyld^−/− mice, respectively. The results present 3 mice of each experimental group.</p

Inhibition of fibrin production abolished protection and increased the hepatic bacterial load of Cyld^−/− mice.

<p>(A) WB analysis of hepatic fibrin production in uninfected and infected WT and Cyld^−/− mice. GAPDH was used as loading control. (B) Quantification of fibrin (± SD) was performed from WB data of uninfected and Lm-infected WT and Cyld^−/−, respectively, which were treated with warfarin as indicated. The results present 3 mice per experimental group. (C) The survival rate of uninfected and infected mice, which were treated with warfarin as indicated, was monitored until day 10 of infection (n = 10 per experimental group). Survival of infected Cyld^−/−, uninfected Cyld^−/− mice treated with warfarin, and WT mice treated with warfarin, respectively, was significantly increased as compared to infected WT mice without warfarin treatment (p<0.001 for all groups). (D) CFUs were determined in the liver of Lm-infected WT and Cyld^−/− mice, which were treated with warfarin as indicated, at day 5 p.i. (* p<0.05, n = 5 per experimental group). Data show the mean ± SD. In (C) and (D) one of two representative experiments is shown.</p