Journal: The Journal of Experimental Medicine

Article Title: Hematopoietic stem cell quiescence and function are controlled by the CYLD–TRAF2–p38MAPK pathway

doi: 10.1084/jem.20141438

Figure Lengend Snippet: CYLD–TRAF2 interaction controls HSC quiescence by inhibiting p38MAPK activation. (A and B) Expression levels of phospho-p38MAPK as determined by flow cytometry in BM HSCs (SKLCD150 + CD48 − CD34 − ) from control and CYLDex7/8 −/− mice after 5-min stimulation with TNF. (C) Cell cycle analysis of BM HSCs isolated from control and CYLDex7/8 −/− mice receiving the p38MAPK inhibitor SB203580 or vehicle (DMSO). (D) Cell cycle analysis of BM HSCs isolated from control and CYLDex7/8 −/− mice receiving the p38MAPK inhibitors LY2228820 and BIRB 796 or vehicle (DMSO). (E and F) Phospho-p38MAPK levels in BM HSCs (SKLCD150 + CD48 − CD34 − ) from control and CYLD Δ932Mx mice. (G) Cell cycle analysis as in C, but analyzing CYLD 932flox/flox (control) and CYLD Δ932Mx (mutant) mice. (H) Cell cycle analysis of BM HSCs isolated from control and CYLD Δ932Mx mice receiving the p38MAPK inhibitors LY2228820 and BIRB 796 or vehicle (DMSO). (I) HSC frequency as determined by flow cytometry in the BM of CYLD 932flox/flox and CYLD Δ932Mx mice receiving SB203580, LY2228820, BIRB 796, or vehicle (DMSO). Results are shown of two (A–D: 4/4; E and F: 3/5; G and H: 5/5; I: 5/5) independent experiments, with the numbers of analyzed control/mutant mice or control/treated groups indicated in parentheses. Error bars indicate SEM. *, P < 0.05; **, P < 0.01.

Article Snippet: After being injected twice with pI-pC, CYLD 932flox/flox and MxCre;CYLD 932flox/flox mice were administered the selective p38MAPK inhibitors SB203580 (50 mg/kg; Selleckchem), LY2228820 (1.5 mg/kg; Selleckchem), BIRB 796 (15 mg/kg; Selleckchem), or vehicle (DMSO).

Techniques: Activation Assay, Expressing, Flow Cytometry, Control, Cell Cycle Assay, Isolation, Mutagenesis

Journal: Biochimica et biophysica acta

Article Title: Calpastatin is upregulated in non-immune neuronal cells via toll-like receptor 2 (TLR2) pathways by lipid-containing agonists.

doi: 10.1016/j.bbamcr.2013.06.006

Figure Lengend Snippet: Fig. 3. LPS activates NF-κB, but does not upregulate calpastatin in SH-SY5Y cells. (A) Cells treated with LPS (50, 100, 1000 ng/ml) for 72 h. Cell extracts were analyzed for calpastatin levels, using tubulin for the estimation of loading. Graph represents means of 2–4 experiments (N = 4 for LPS 50 ng/ml; N = 2 for LPS 100 and 1000 ng/ml); % of control denotes the calpastatin levels versus the control (considered 100%). (B) NF-κB in the cell nuclei. Cells were cultured in the presence and absence of LPS (100 ng/ml); cell extracts were an- alyzed for nuclear levels of p65 and p50 subunits, using the lamin A band for the estimation of loading. Graph represents means ± S.E.M. (N = 3–4) **P b 0.01 versus control, untreated cells (considered 100%). (C)-(G) Cells pretreated with inhibitors, then LPS (100 ng/ml) added; cell nuclear extracts were analyzed for nuclear levels of p65 subunit. Lamin A, B23, or Histon 3 (H3) were used for the estimation of loading. (C) Cells pretreated with (5Z)-7-Oxozeaenol (300 μM). Graph represents means ± S.E.M. (N = 4) **P b 0.01 versus control cells (considered 100%); ##P b 0.01 versus LPS-treated cells. (D) Cells pretreated with PS1145 (10 μM). Graph represents means ± S.E.M. (N = 3) *P b 0.05, **P b 0.01 versus control, untreated cells (considered 100%). (E) Cells pretreated with PD98059 (10 μM). Graph represents an average of two experiments. (F) Cells pretreated with SB203580 (1 μM). Graph represents means ± S.E.M. (N = 3); **P b 0.01 versus control, untreated cells (considered 100%). (G) Cells pretreated with TAT-DEF-ELK1 (10 μM). Graph represents an average of two experiments. C, control; OZ, (5Z)-7-Oxozeaenol; PS, PS1145; PD, PD98059; SB, SB203580; TDE, TAT-DEF-ELK1.

Article Snippet: The inhibitors used were: Anti TLR-2 antibody (αTLR) (N-17 (Sc-8689, Santa Cruz Biotechnology, Santa Cruz, CA, USA)) (stock solution of 200 μg/ml) was added at a final concentration of 6.6 μg/ml; the TAK1 inhibitor (5Z)-7-Oxozeaenol (499610, Calbiochem) (stock solution of 300 μM)was added at a final concentration of 300 nM; the IKK inhibitor PS1145 (Sc-301621, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (stock solution of 10 mM) was added at a final concentration of 10 μM; the MEK 1/2 selective inhibitor PD98059 (513000, Calbiochem, MERCK, Darmstadt, Germany) (stock solution of 10 mM) was added at a final concentration of 10 μM; The synthetic cell penetrating peptide TATDEF-ELK1 (TDE) that inhibits ERK-induced ELK1 phosphorylation [20] (a generous gift from J. Caboche, Université Pierre et Marie Curie, Paris, France, patents EP 05 290 363 and WO 2006/087242) (stock solution of 1 mM)was added at a final concentration of 10 μM; the p38MAPK inhibitor SB203580 (5633, Cell Signaling Technology, MA, USA) (stock solution of 30 mM) was added at a final concentration of 1 μM.

Techniques: Control, Cell Culture

Journal: Biochimica et biophysica acta

Article Title: Calpastatin is upregulated in non-immune neuronal cells via toll-like receptor 2 (TLR2) pathways by lipid-containing agonists.

doi: 10.1016/j.bbamcr.2013.06.006

Figure Lengend Snippet: Fig. 4. Lipoteichoic acid (LTA) upregulates calpastatin, but does not activates NF-κB in SH-SY5Y cells. (A) Cultured cells were treated with LTA (100 ng/ml) for 72 h; nuclear extracts were analyzed for levels of p65 and p50 subunits, using the B23 band for the estimation of loading. Graph represents means ± S.E.M. (N = 2–3). (B) Cultured cells were treated with LTA (100 ng/ml) for 24-72 h; cell extracts were analyzed for calpastatin levels. Graph represents means ± S.E.M. (N = 3; for 24 h, average of two experiments). *P b 0.05, ***P b 0.001 versus control, untreated cells (considered 100%). (C–E) cells pretreated for 1 h with vehicle and with inhibitors, and cultures continued for 72 h without and with LTA (100 ng/ml); cell extracts were analyzed for calpastatin levels. (C) Cells pretreated with PD98059 (10 μM). Graph represents means ± S.E.M. (N = 3); ***P b 0.001 versus con- trol, untreated cells (considered 100%); ###P b 0.001 versus LTA-treated cells. (D) Cells pretreated with (5Z)-7-Oxozeaenol (300 μM). Graph represents means ± S.E.M. (N = 3); **P b 0.01, ***P b 0.001 versus control, untreated cells (considered 100%); (E) Cells pretreated with PS1145 (10 μM). Graph represents an average of two experiments. (F) Cells pretreated with TAT-DEF-ELK1. Graph represents means ± S.E.M. (N = 4); ***P b 0.001 versus control, untreated cells (considered 100%); ###P b 0.001 versus LTA-treated cells. (G) Cells pretreated with SB203580 (1 μM). Graph represents an average of two experiments. C, control; PD, PD98059; OZ, (5Z)-7-Oxozeaenol; PS, PS1145; TDE, TAT-DEF-ELK1; Clps, calpastatin; Tubl, tubulin.

Article Snippet: The inhibitors used were: Anti TLR-2 antibody (αTLR) (N-17 (Sc-8689, Santa Cruz Biotechnology, Santa Cruz, CA, USA)) (stock solution of 200 μg/ml) was added at a final concentration of 6.6 μg/ml; the TAK1 inhibitor (5Z)-7-Oxozeaenol (499610, Calbiochem) (stock solution of 300 μM)was added at a final concentration of 300 nM; the IKK inhibitor PS1145 (Sc-301621, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (stock solution of 10 mM) was added at a final concentration of 10 μM; the MEK 1/2 selective inhibitor PD98059 (513000, Calbiochem, MERCK, Darmstadt, Germany) (stock solution of 10 mM) was added at a final concentration of 10 μM; The synthetic cell penetrating peptide TATDEF-ELK1 (TDE) that inhibits ERK-induced ELK1 phosphorylation [20] (a generous gift from J. Caboche, Université Pierre et Marie Curie, Paris, France, patents EP 05 290 363 and WO 2006/087242) (stock solution of 1 mM)was added at a final concentration of 10 μM; the p38MAPK inhibitor SB203580 (5633, Cell Signaling Technology, MA, USA) (stock solution of 30 mM) was added at a final concentration of 1 μM.

Techniques: Cell Culture, Control

Journal:

Article Title: Involvement of multiple signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy

doi: 10.1073/pnas.1137463100

Figure Lengend Snippet: (a) Growth inhibition of lymphoma cell lines in response to blockade of p38MAPK activity. Growth inhibition was measured by a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide conversion at 6, 24, and 48 h in comparison to DMSO-treated control cells. The results are expressed as inhibition of growth compared with control DMSO-treated cells and represent the mean ± SD of the results obtained in triplicate. Blockade of p38MAPK activity resulted in significant dose-dependent and time-dependent growth inhibition in OCI LY-1 and SUDHL-4 cells, which are t(14;18)+/p38MAPK+ (Upper). By contrast, the t(14;18)-negative/p38MAPK-negative cell line SUDHL-5 was relatively unaffected by similar doses of SB203580 (Lower Left). The specificity of p38MAPK as a target was confirmed by the lack growth inhibition by U0126 the p42/44MAPK inhibitor in the t(14;18)+/p38+ SUDHL-4 cell line (Lower Right). One representative experiment of six independent repetitions is illustrated. (b) SB203580 treatment induces caspase-3 activity. Treatment of SUDHL-4 cells with 30 μM SB203580 resulted in significant time-dependent induction of caspase-3 activity (12.6% at 6 h and 41% at 24 h; P = 0.02). Caspase-3 induction was inhibited by Z-VAD-FMK, a selective caspase-3 inhibitor. One representative experiment of three independent repetitions is illustrated. (c) Inhibition of p38MAPK induces apoptosis in t(14;18)+ cell lines. Cells were cultured in the presence of DMSO or SB203580 (50 μM) and stained for annexin V binding at the times indicated. The control represents DMSO-treated cells at 72 h. (Right) We illustrate a representative histogram for t(14;18) cell lines cultured in the presence of DMSO or SB203580 at the indicated concentrations for 72 h. Apoptosis was determined by annexin V binding and expressed as net apoptosis induction [percentage of apoptosis in treated cells minus percentage of apoptosis in control (DMSO-treated) cells]. One representative experiment of two independent repetitions is illustrated. (d) SB203580 results in selective inhibition of p38MAPK activation. Western blot analysis revealed high basal levels of phosphorylated p38MAPK in SUDHL-4 cells. Treatment with 30 μM SB203580 resulted in a significant decrease in the level of phospho-p38MAPK at 6 h. The levels of total p38MAPK and total and phospho-p42/p44 MAPK were unaffected by SB203580 treatment.

Article Snippet: SUDHL-4, SUDHL-5, and OCI-LY1 cell lines (1.5 × 10 5 cells per ml) were treated with SB203580, a selective p38MAPK inhibitor (Upstate Biotechnology, Lake Placid, NY), or U0126, a selective p42/44MAPK inhibitor (Cell Signaling, Beverly, MA) dissolved in DMSO.

Techniques: Inhibition, Activity Assay, Cell Culture, Staining, Binding Assay, Activation Assay, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Hematopoietic stem cell quiescence and function are controlled by the CYLD–TRAF2–p38MAPK pathway

doi: 10.1084/jem.20141438

Figure Lengend Snippet: CYLD–TRAF2 interaction controls HSC quiescence by inhibiting p38MAPK activation. (A and B) Expression levels of phospho-p38MAPK as determined by flow cytometry in BM HSCs (SKLCD150 + CD48 − CD34 − ) from control and CYLDex7/8 −/− mice after 5-min stimulation with TNF. (C) Cell cycle analysis of BM HSCs isolated from control and CYLDex7/8 −/− mice receiving the p38MAPK inhibitor SB203580 or vehicle (DMSO). (D) Cell cycle analysis of BM HSCs isolated from control and CYLDex7/8 −/− mice receiving the p38MAPK inhibitors LY2228820 and BIRB 796 or vehicle (DMSO). (E and F) Phospho-p38MAPK levels in BM HSCs (SKLCD150 + CD48 − CD34 − ) from control and CYLD Δ932Mx mice. (G) Cell cycle analysis as in C, but analyzing CYLD 932flox/flox (control) and CYLD Δ932Mx (mutant) mice. (H) Cell cycle analysis of BM HSCs isolated from control and CYLD Δ932Mx mice receiving the p38MAPK inhibitors LY2228820 and BIRB 796 or vehicle (DMSO). (I) HSC frequency as determined by flow cytometry in the BM of CYLD 932flox/flox and CYLD Δ932Mx mice receiving SB203580, LY2228820, BIRB 796, or vehicle (DMSO). Results are shown of two (A–D: 4/4; E and F: 3/5; G and H: 5/5; I: 5/5) independent experiments, with the numbers of analyzed control/mutant mice or control/treated groups indicated in parentheses. Error bars indicate SEM. *, P < 0.05; **, P < 0.01.

Article Snippet: After being injected twice with pI-pC, CYLD 932flox/flox and MxCre;CYLD 932flox/flox mice were administered the selective p38MAPK inhibitors SB203580 (50 mg/kg; Selleckchem), LY2228820 (1.5 mg/kg; Selleckchem), BIRB 796 (15 mg/kg; Selleckchem), or vehicle (DMSO).

Techniques: Activation Assay, Expressing, Flow Cytometry, Control, Cell Cycle Assay, Isolation, Mutagenesis

Journal: Biochimica et biophysica acta

Article Title: Calpastatin is upregulated in non-immune neuronal cells via toll-like receptor 2 (TLR2) pathways by lipid-containing agonists.

doi: 10.1016/j.bbamcr.2013.06.006

Figure Lengend Snippet: Fig. 1. Inhibition of LPP-induced calpastatin upregulation in SH-SY5Y cells. Cultured cells were pretreated for 1 h with inhibitors, and cultures continued for 72 h in the presence of lipoproteins (0.3 μg/ml). Calpastatin levels were determined by western blotting, as described in Materials and methods. Tubulin was used for the estimation of loading; % of control denotes the calpastatin levels. (A) Cells pretreated with anti-TLR2 antibody (6.6 μg/ml). Graph represents means ± S.E.M. (N = 5) ***P b 0.001 versus control cells (considered 100%); ##P b 0.01 versus LPP-treated cells. (B) Cells pretreated with (5Z)-7-Oxozeaenol (300 nM). Graph represents means ± S.E.M. (N = 4) **P b 0.01 versus control cells (con- sidered 100%); ##P b 0.01 versus LPP-treated cells. (C) Cells pretreated with PS1145 (10 μM). Graph represents means ± S.E.M. (N = 3; for PS1145 alone, average of two exper- iments). **P b 0.01 versus control cells (considered 100%); ##P b 0.01 versus LPP-treated cells. C, control; LPP, lipoproteins; αTLR, anti-TLR2 antibody; OZ, (5Z)-7-Oxozeaenol; PS, PS1145; Clps, calpastatin; Tubl, tubulin.

Article Snippet: The inhibitors used were: Anti TLR-2 antibody (αTLR) (N-17 (Sc-8689, Santa Cruz Biotechnology, Santa Cruz, CA, USA)) (stock solution of 200 μg/ml) was added at a final concentration of 6.6 μg/ml; the TAK1 inhibitor (5Z)-7-Oxozeaenol (499610, Calbiochem) (stock solution of 300 μM)was added at a final concentration of 300 nM; the IKK inhibitor PS1145 (Sc-301621, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (stock solution of 10 mM) was added at a final concentration of 10 μM; the MEK 1/2 selective inhibitor PD98059 (513000, Calbiochem, MERCK, Darmstadt, Germany) (stock solution of 10 mM) was added at a final concentration of 10 μM; The synthetic cell penetrating peptide TATDEF-ELK1 (TDE) that inhibits ERK-induced ELK1 phosphorylation [20] (a generous gift from J. Caboche, Université Pierre et Marie Curie, Paris, France, patents EP 05 290 363 and WO 2006/087242) (stock solution of 1 mM)was added at a final concentration of 10 μM; the p38MAPK inhibitor SB203580 (5633, Cell Signaling Technology, MA, USA) (stock solution of 30 mM) was added at a final concentration of 1 μM.

Techniques: Inhibition, Cell Culture, Western Blot, Control

Journal: Biochimica et biophysica acta

Article Title: Calpastatin is upregulated in non-immune neuronal cells via toll-like receptor 2 (TLR2) pathways by lipid-containing agonists.

doi: 10.1016/j.bbamcr.2013.06.006

Figure Lengend Snippet: Fig. 3. LPS activates NF-κB, but does not upregulate calpastatin in SH-SY5Y cells. (A) Cells treated with LPS (50, 100, 1000 ng/ml) for 72 h. Cell extracts were analyzed for calpastatin levels, using tubulin for the estimation of loading. Graph represents means of 2–4 experiments (N = 4 for LPS 50 ng/ml; N = 2 for LPS 100 and 1000 ng/ml); % of control denotes the calpastatin levels versus the control (considered 100%). (B) NF-κB in the cell nuclei. Cells were cultured in the presence and absence of LPS (100 ng/ml); cell extracts were an- alyzed for nuclear levels of p65 and p50 subunits, using the lamin A band for the estimation of loading. Graph represents means ± S.E.M. (N = 3–4) **P b 0.01 versus control, untreated cells (considered 100%). (C)-(G) Cells pretreated with inhibitors, then LPS (100 ng/ml) added; cell nuclear extracts were analyzed for nuclear levels of p65 subunit. Lamin A, B23, or Histon 3 (H3) were used for the estimation of loading. (C) Cells pretreated with (5Z)-7-Oxozeaenol (300 μM). Graph represents means ± S.E.M. (N = 4) **P b 0.01 versus control cells (considered 100%); ##P b 0.01 versus LPS-treated cells. (D) Cells pretreated with PS1145 (10 μM). Graph represents means ± S.E.M. (N = 3) *P b 0.05, **P b 0.01 versus control, untreated cells (considered 100%). (E) Cells pretreated with PD98059 (10 μM). Graph represents an average of two experiments. (F) Cells pretreated with SB203580 (1 μM). Graph represents means ± S.E.M. (N = 3); **P b 0.01 versus control, untreated cells (considered 100%). (G) Cells pretreated with TAT-DEF-ELK1 (10 μM). Graph represents an average of two experiments. C, control; OZ, (5Z)-7-Oxozeaenol; PS, PS1145; PD, PD98059; SB, SB203580; TDE, TAT-DEF-ELK1.

Article Snippet: The inhibitors used were: Anti TLR-2 antibody (αTLR) (N-17 (Sc-8689, Santa Cruz Biotechnology, Santa Cruz, CA, USA)) (stock solution of 200 μg/ml) was added at a final concentration of 6.6 μg/ml; the TAK1 inhibitor (5Z)-7-Oxozeaenol (499610, Calbiochem) (stock solution of 300 μM)was added at a final concentration of 300 nM; the IKK inhibitor PS1145 (Sc-301621, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (stock solution of 10 mM) was added at a final concentration of 10 μM; the MEK 1/2 selective inhibitor PD98059 (513000, Calbiochem, MERCK, Darmstadt, Germany) (stock solution of 10 mM) was added at a final concentration of 10 μM; The synthetic cell penetrating peptide TATDEF-ELK1 (TDE) that inhibits ERK-induced ELK1 phosphorylation [20] (a generous gift from J. Caboche, Université Pierre et Marie Curie, Paris, France, patents EP 05 290 363 and WO 2006/087242) (stock solution of 1 mM)was added at a final concentration of 10 μM; the p38MAPK inhibitor SB203580 (5633, Cell Signaling Technology, MA, USA) (stock solution of 30 mM) was added at a final concentration of 1 μM.

Techniques: Control, Cell Culture

Journal: Biochimica et biophysica acta

Article Title: Calpastatin is upregulated in non-immune neuronal cells via toll-like receptor 2 (TLR2) pathways by lipid-containing agonists.

doi: 10.1016/j.bbamcr.2013.06.006

Figure Lengend Snippet: Fig. 4. Lipoteichoic acid (LTA) upregulates calpastatin, but does not activates NF-κB in SH-SY5Y cells. (A) Cultured cells were treated with LTA (100 ng/ml) for 72 h; nuclear extracts were analyzed for levels of p65 and p50 subunits, using the B23 band for the estimation of loading. Graph represents means ± S.E.M. (N = 2–3). (B) Cultured cells were treated with LTA (100 ng/ml) for 24-72 h; cell extracts were analyzed for calpastatin levels. Graph represents means ± S.E.M. (N = 3; for 24 h, average of two experiments). *P b 0.05, ***P b 0.001 versus control, untreated cells (considered 100%). (C–E) cells pretreated for 1 h with vehicle and with inhibitors, and cultures continued for 72 h without and with LTA (100 ng/ml); cell extracts were analyzed for calpastatin levels. (C) Cells pretreated with PD98059 (10 μM). Graph represents means ± S.E.M. (N = 3); ***P b 0.001 versus con- trol, untreated cells (considered 100%); ###P b 0.001 versus LTA-treated cells. (D) Cells pretreated with (5Z)-7-Oxozeaenol (300 μM). Graph represents means ± S.E.M. (N = 3); **P b 0.01, ***P b 0.001 versus control, untreated cells (considered 100%); (E) Cells pretreated with PS1145 (10 μM). Graph represents an average of two experiments. (F) Cells pretreated with TAT-DEF-ELK1. Graph represents means ± S.E.M. (N = 4); ***P b 0.001 versus control, untreated cells (considered 100%); ###P b 0.001 versus LTA-treated cells. (G) Cells pretreated with SB203580 (1 μM). Graph represents an average of two experiments. C, control; PD, PD98059; OZ, (5Z)-7-Oxozeaenol; PS, PS1145; TDE, TAT-DEF-ELK1; Clps, calpastatin; Tubl, tubulin.

Article Snippet: The inhibitors used were: Anti TLR-2 antibody (αTLR) (N-17 (Sc-8689, Santa Cruz Biotechnology, Santa Cruz, CA, USA)) (stock solution of 200 μg/ml) was added at a final concentration of 6.6 μg/ml; the TAK1 inhibitor (5Z)-7-Oxozeaenol (499610, Calbiochem) (stock solution of 300 μM)was added at a final concentration of 300 nM; the IKK inhibitor PS1145 (Sc-301621, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (stock solution of 10 mM) was added at a final concentration of 10 μM; the MEK 1/2 selective inhibitor PD98059 (513000, Calbiochem, MERCK, Darmstadt, Germany) (stock solution of 10 mM) was added at a final concentration of 10 μM; The synthetic cell penetrating peptide TATDEF-ELK1 (TDE) that inhibits ERK-induced ELK1 phosphorylation [20] (a generous gift from J. Caboche, Université Pierre et Marie Curie, Paris, France, patents EP 05 290 363 and WO 2006/087242) (stock solution of 1 mM)was added at a final concentration of 10 μM; the p38MAPK inhibitor SB203580 (5633, Cell Signaling Technology, MA, USA) (stock solution of 30 mM) was added at a final concentration of 1 μM.

Techniques: Cell Culture, Control