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

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

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 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