16 research outputs found

    Nec-1 blocks sustained phosphorylation of LAT during T cell activation.

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    <p>(A) Whole cell extracts (WCE) were examined from T cells stimulated with anti-CD3 antibody in the presence or absence of 50 µM Nec-1. Western blot was performed with phospho-tyrosine specific antibody. Short and long exposures were shown to highlight the different phosphorylated species. (B) Nec-1 impairs TCR-induced LAT phosphorylation. Total lysates from activated T cells were subjected to immunoprecipitation with phospho-tyrosine antibody. The resulting immune complex was evaluated for presence of LAT and Lck by Western blot. (C) Nec-1 does not affect Erk phosphorylation. Purified T cells were stimulated as in (A). Western blot was performed with phospo-Erk or total Erk specific antibodies.</p

    Knock-down of RIP1 expression in L929 cells did not result in caspase-dependent apoptosis.

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    <p>L929 cells were transfected with siRNA against (A) RIP1 or (B) TR4. Transfected cells were treated with zVAD-fmk, Nec-1 and TNF as indicated. Cell death was determined by MTS assay. (C) L929 cells transfected with the indicated siRNA or Jurkat 4E3 cells were treated with TNF. PARP-1 cleavage was determined by Western blot. (D) Cell lysates from (C) were tested for active caspase 3 as measured by cleavage of the substrate DEVD-AMC. Release of AMC was determined by increases in AMC fluorescence as described in materials and methods.</p

    Nec-1 inhibits necrosis in L929 cells in a dose-dependent manner.

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    <p>(A) Nec-1 inhibits TNF-induced necrosis in L929 cells. L929 cells were treated with 10 µM zVAD-fmk, 20 µM Nec-1 and 10 ng/ml mouse TNF (mTNF) as indicated. Cell death was determined by MTS assay as described in materials and methods. (B) L929 cells were treated with the indicated doses of Nec-1, followed by stimulation with 10 ng/ml mTNF. Cell Death was analyzed as in (A). (C) Nec-1 did not alter RIP1 or RIP3 protein expression in L929 cells.</p

    High doses of Nec-1 inhibit T cell proliferation.

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    <p>(A) Low dose Nec-1 inhibits TCR-induced necrosis, but high dose Nec-1 inhibits T cell proliferation. Purified T cells from wild type or FADD<sup>−/−</sup> mice were stimulated with plate-bound anti-CD3, anti-CD28 and increasing doses of Nec-1. Three days later, cell proliferation was measured by incorporation of [<sup>3</sup>H]-thymidine. Results shown are mean ± SEM. (B) Nec-1 did not compromised T cell viability. Naïve CD3<sup>+</sup> T cells were purified from the spleen of wild type C57BL/6 mice and incubated at 37°C for 24 hours in the presence or absence of 50 µM Nec-1. Viability of the cells was determined by flow cytometry using propidium iodide (PI) uptake as an indication of cell death. Note that Nec-1 increased the baseline fluorescence of the T cells. (C) Nec-1 inhibits T cell division. Purified CD3<sup>+</sup> primary T cells were labeled with CellTracer Violet fluorescent dye and stimulated with 1 µg/ml plate-bound anti-CD3 and 200 ng/ml anti-CD28 antibodies. Three days later, cell division was analyzed using a BD LSR2 flow cytometer. The numbers above each peak represent the number of cell division the cells had undergone. The numbers on the left represent the percentages of cells in each peak. (D) Nec-1 inhibits T cell blast formation. Purified CD3<sup>+</sup> T cells were similarly activated as in (C). Three days later, formation of T cell blast as measured by forward scatter was determined by flow cytometry. (E) FADD<sup>−/−</sup>RIP1<sup>−/−</sup> DKO T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the absence or presence of Nec-1 were measured for cell proliferation as in (C).</p

    Effects of Nec-1 on Jnk, Erk and PKA-Cβ.

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    <p>(A) L929 cells were treated with TNF in the presence of 20 µM Nec-1 or 50 µM of the Erk inhibitor U0126. Cell death was determined by MTS assay. (B) The effect of Nec-1 on Erk or Jnk phosphorylation. L929 cells treated with 10 ng/ml of mTNF in the presence or absence of 30 µM Nec-1 were analyzed for Erk activation (p-p54 and p-p46) or Jnk activation by Western blot. (C) Erk inhibitor U0126 and Nec-1 blocks TNF-induced necrosis in Jurkat cells. Jurkat 4E3 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023209#pone.0023209-Chan2" target="_blank">[20]</a> were treated with Erk inhibitor (50 µM U0126), Jnk inhibitor (30 µM Jnk inhibitor II), p38 inhibitor (50 µM SB203580), or 10 µM Nec-1 and stimulated with 100 ng/ml recombinant human TNF plus 10 µM zVAD-fmk for 14 hours. Cell death was determined by MTS assay. (D) Jurkat 4E3 cells were stimulated with 10 ng/ml PMA for 10 minutes in the presence of 30 µM Nec-1 or 50 µM U0126. Phospho-Erk was examined by Western blot as indicated. (E) Jurkat 4E3 cells were treated with the indicated doses of Nec-1 and 100 nM calyculin A. The expression of the two different PKA isoforms and phosphorylation of protein kinase A substrates was determined by Western blot with specific μantibodies as described in materials and methods.</p

    Nec-1 inhibits TNF-induced necrosis independent of RIP1 in L929 cells.

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    <p>(A–C) RIP1 is required for zVAD-fmk, but not TNF-induced necrosis in L929 cells. (A) L929 cells were transfected with the indicated siRNA oligonucleotides with HiPerfect (Qiagen). TR4 is the control siRNA against TRAIL-R4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023209#pone.0023209-Clancy1" target="_blank">[36]</a>. Cells were treated with (A) 50 µM zVAD-fmk or (B) 10 ng/ml mTNF and cell death was measured by MTS assay. (C) Western blot shows RIP1 and RIP3 expression in cells transfected with the indicated siRNAs. (D–E) RIP1 is required for TNF-induced necrosis in 3T3 fibroblasts. NIH 3T3 fibroblasts were transfected with the indicated siRNA. (D) Expression of RIP1, RIP3 and ß-actin in cells transfected with the indicated siRNA was determined by Western blot. (E) Necrosis was induced with 10 µM zVAD-fmk, 0.5 µg/ml cycloheximide and 10 ng/ml mTNF for 20 hours. Cell death was assessed as in (A).</p

    The effect of p38α inhibition or deficiency on WAT browning is cell autonomous.

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    <p>(A-C) Representative HE staining of iWAT (A), diameter and cross-sectional area of adipocytes in iWAT (B), and representative UCP-1 staining of iWAT (C) from C57BL/6J mice after injection of Ad-p38αAF into the fat pad of iWAT. These mice were exposed to cold for 2 d before analysis. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (D-F) Relative mRNA levels of UCP-1, PGC1α, DIO2, COX8B, and/or ELVOL3 in iWAT-SVF-derived matured adipocytes treated with SB203580 for 4 or 8 h (D, <i>n</i> = 4 per group), or p38α-specific inhibitor (p38αMAPK-IN-1) for 4 h (E, <i>n</i> = 4 per group), or infected with Lenti-p38αAF (F, <i>n</i> = 3 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (G) Relative mRNA levels of UCP-1, PGC1α, PRDM16, DIO2, ELVOL3, COX8B, and CIDEA in matured adipocytes derived from iWAT-SVF of Floxed and Fp38αKO mice (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (H) Representative fluorescence staining of Mito in matured iWAT-SVF-derived adipocytes infected with Lenti-p38αAF. Bars: 25 μm. (I and J) OCR of Oligomycin, FCCP, and Antimycin A/Rotenone-treated matured adipocytes derived from iWAT-SVF of Floxed and Fp38αKO mice (I) and the AUC of OCR (J) as indicated (<i>n</i> = 5 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (K and L) Representative UCP-1 staining of iWAT-SVF-derived matured adipocytes after infection with Lenti-p38αAF (K) or matured adipocytes derived from iWAT-SVF of Floxed and Fp38αKO mice (L). Bars: 10 μm. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Ad-p38αAF, adenovirus expressing p38αAF; AUC, area under the curve; CIDEA, cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; COX8B, cytochrome c oxidase subunit 8B; DIO2, deiodinase 2; ELVOL3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; HE staining, hematoxylin-eosin staining; IHC, immunohistochemistry; iWAT, inguinal white adipose tissue; Lenti-p38αAF, lentivirus expressing p38αAF; MAPK, mitogen-activated protein kinase; Mito, mitochondria; NS, not significant; OCR, oxygen consumption rate; PGC1α, peroxisome proliferative activated receptor gamma coactivator 1α; PRDM16, positive regulatory domain containing 16; SVF, stromal vascular fraction; UCP-1, uncoupling protein 1; WAT, white adipose tissue.</p

    Loss of p38α in adipose tissues causes minimal effects on BAT.

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    <p>(A) BT of Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 6 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (B and C) VO<sub>2</sub> (B) and VCO<sub>2</sub> (C) in Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 4 per group). The values were normalized by LM. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (D) iBAT weight and relative iBAT weight to BW ratio (iBAT/BW) of Floxed (<i>n</i> = 10) and Fp38αKO (<i>n</i> = 8). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (E-G) Representative HE staining of iBAT (E), diameter and cross-sectional area of adipocytes in iBAT (F), and representative UCP-1 staining of iBAT (G) from Floxed and Fp38αKO mice maintained at RT. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (H) Representative EM images of iBAT from Floxed and Fp38αKO mice maintained at RT at low (top), medium (middle), and high (bottom) magnification, as indicated. (I) Relative mitDNA to nuDNA ratio in unilateral iBAT of Floxed (<i>n</i> = 5) and Fp38αKO (<i>n</i> = 9) mice maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (J) OCR of ADP, Oligomycin, FCCP, and Antimycin A/Rotenone-treated mitochondria derived from iBAT of Floxed and Fp38αKO mice exposed to cold for 2 d (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (K) Relative mRNA levels of UCP-1, PGC1α, DIO2, COX8B, and PRDM16 in iBAT from Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 6 per group) or exposed to cold for 2 d (<i>n</i> = 8 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (L and M) Representative western blots of UCP-1 in iBAT from Floxed and Fp38αKO mice maintained at RT (L) or exposed to cold for 2 d (M). (N) BT of Fp38αKO and Floxed mice exposed to cold for 4 h (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (O) Relative mRNA levels of UCP-1 and PGC1α in iBAT from Floxed (<i>n</i> = 7–8) and Fp38αKO (<i>n</i> = 8) mice exposed to cold for 4 h. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (P and Q) Representative western blots (P) and densitometry analysis (Q) of UCP-1 in iBAT from Floxed and Fp38αKO mice exposed to cold for 4 h. The densities of UCP-1 bands were quantitated and normalized to Hsp90 (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (R and S) Relative mRNA levels of PRDM16, DIO2, ELVOL3, COX8B, and CIDEA(R), ATGL, MGL, and HSL (S) in iBAT from Floxed (<i>n</i> = 7–8) and Fp38αKO (<i>n</i> = 6–8) mice exposed to cold for 4 h. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. ADP, adenosine diphosphate; ATGL, adipose triglyceride lipase; BAT, brown adipose tissue; BT, body temperature; BW, body weight; CIDEA, cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; COX8B, cytochrome c oxidase subunit 8B; DIO2, deiodinase 2; ELVOL3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; EM, electron microscopy; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; HE staining, hematoxylin-eosin staining; HSL, hormone-sensitive lipase; iBAT, interscapular brown adipose tissue; IHC, immunohistochemistry; LM, lean mass; MGL, monoglyceride lipase; mitDNA, mitochondrial DNA; NS, not significant; nuDNA, nuclear DNA; OCR, oxygen consumption rate; PGC1α, peroxisome proliferative activated receptor gamma coactivator 1α; PRDM16, positive regulatory domain containing 16; RT, room temperature; UCP-1, uncoupling protein 1; VCO<sub>2</sub>, carbon dioxide production; VO<sub>2</sub>, oxygen consumption.</p

    Adipocyte-specific deletion of p38α leads to a lean phenotype and increased glucose tolerance and insulin sensitivity.

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    <p>(A-C) Representative western blots of p38α and p-p38 in iBAT (A), iWAT (B), and eWAT (C) from Floxed and Fp38αKO mice as indicated. (D-F) Representative western blots of p38α in other tissues, including liver (D), skeletal muscle (E), and macrophages (F) from Floxed and Fp38αKO mice as indicated. (G) Cumulative gross energy intake and feces of Floxed and Fp38αKO mice for 24 h (<i>n</i> = 4 per group). Mice were maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (H) Growth curve of Floxed (<i>n</i> = 15) and Fp38αKO (<i>n</i> = 8–13) mice maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (I and J) BW, FM, and FM to BW ratio (FM/BW) of Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 11 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (K and L) GTT (K, <i>n</i> = 8 per group) and ITT (L, <i>n</i> = 8 per group) in Floxed and Fp38αKO mice. AUCs were calculated. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01. AUC, area under curve; BW, body weight; eWAT, epididymal white adipose tissue; FM, fat mass; GTT, glucose tolerance test; iBAT, interscapular brown adipose tissue; ITT, insulin tolearance test; iWAT, inguinal white adipose tissue; NS, not significant; RT, room temperature.</p

    Pharmaceutical inhibition of p38α reduces adiposity and enhances the browning of WAT.

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    <p>(A and B) Representative HE staining of iBAT, iWAT, and eWAT (A), diameter and cross-sectional area (B) of adipocytes in these adipose tissues from SB203580-treated C57BL/6J mice at 2 d postinjection. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (C) BW of C57BL/6J mice before and after 4 wk of SB203580 treatment (control: <i>n</i> = 4, SB203580: <i>n</i> = 5). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (D and E) Relative weight of iWAT and eWAT from C57BL/6J mice after 4 wk of SB203580 treatment. These mice were maintained at RT (D, <i>n</i> = 5 per group) or exposed to cold for 2 d before analysis (E, <i>n</i> = 5 per group) as indicated. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (F) Relative mRNA levels of UCP-1, PGC1α, ELOVL3, and DIO2 in iWAT from C57BL/6J mice after 4 wk of SB203580 treatment (<i>n</i> = 10 per group). These mice were exposed to cold for 2 d before analysis. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (G) Representative western blots of p-CREB (Ser133) and UCP-1 in iWAT from C57BL/6J mice after 4 wk of SB203580 treatment. These mice were exposed to cold for 2 d before analysis. (H and I) Representative PET/CT images (H) and PET images (I) of C57BL/6J mice after 4 wk of SB203580 treatment. These mice received a daily CL316,243 injection for 8 d before <sup>18</sup>F-FDG injection. White dashed triangles represent the anatomical sites of iWAT. (J) Ex vivo measured <sup>18</sup>F-FDG uptake in iBAT, iWAT, and eWAT to tissue weight ratio by γ counter (<i>n</i> = 3 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (K) Relative weight of iWAT from <i>db/db</i> mice after 3 wk of SB203580 treatment (<i>n</i> = 5 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (L and M) Representative HE staining of iWAT (L), diameter and cross-sectional area of adipocytes in iWAT (M) from <i>db/db</i> mice after 3 wk of SB203580 treatment. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. BW, body weight; CREB, cAMP-response element binding protein; CT, computed tomography; DIO2, deiodinase 2; ELVOL3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; eWAT, epididymal white adipose tissue; HE staining, hematoxylin-eosin staining; iBAT, interscapular brown adipose tissue; iWAT, inguinal white adipose tissue; NS, not significant; PET, positron emission tomography; PGC1α, peroxisome proliferative activated receptor gamma coactivator 1α; RT, room temperature; UCP-1, uncoupling protein 1; WAT, white adipose tissue.</p
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