Journal: Arthritis and rheumatism

Article Title: Leishmanial lipid suppresses tumor necrosis factor alpha, interleukin-1beta, and nitric oxide production by adherent synovial fluid mononuclear cells in rheumatoid arthritis patients and induces apoptosis through the mitochondrial-mediated pathway.

doi: 10.1002/art.23295

Figure Lengend Snippet: Figure 5. A, Dose kinetics of leishmanial lipid–induced activation of caspase 3, caspase 8, and caspase 9 in adherent SFMCs. Cells were treated with different concentrations of leishmanial lipid for 48 hours. Cell extracts were prepared, and activity levels of caspase 3, caspase 8, and caspase 9 were measured by colorimetric assay as described in Materials and Methods. B, Effects of inhibitors of caspase 3 (Z-DEVD-FMK), caspase 8 (Z-IETD-FMK), and caspase 9 (Z-LEHD-FMK) on leishmanial lipid– induced apoptosis of adherent SFMCs. Cells were pretreated with 30 M specific caspase inhibitors for 1 hour, and apoptosis was assayed by fluorescence-activated cell sorting. C and D, Effects of leishmanial lipid on cytosolic cytochrome c levels (C) and mitochondrial membrane potential (D). Cells treated with different concentrations of leishmanial lipid for the indicated time periods were processed as described in Materials and Methods. The level of cytosolic cytochrome c was measured by enzyme-linked immunosor- bent assay. Rhodamine fluorescence was assayed by spectrofluorometry and used as a measure of mitochondrial transmembrane potential. Values are the mean SEM from 1 of 3 representative experiments. P 0.001 versus untreated cells. OD optical density; AU arbitrary units (see Figure 2 for other definitions).

Article Snippet: Antibodies to IL-10, TNF , IL-1 , inducible nitric oxide synthase (iNOS), caspase 3, caspase 8, and caspase 9, and cytochrome c, -actin, alkaline phosphatase (AP)/horseradish peroxidase–conjugated IgG, Z-DEVD-FMK, Z-LEHD-FMK, and Z-IETD-FMK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Activity Assay, Colorimetric Assay, Fluorescence, FACS, Membrane

Journal: Cell Death & Disease

Article Title: SGK-1 protects kidney cells against apoptosis induced by ceramide and TNF- α

doi: 10.1038/cddis.2015.232

Figure Lengend Snippet: Apoptosis induced by TNF- α is mediated via caspase-3 activation. HEK-293 cells were transfected with Mock, SGK-1wt and SGK-1dn constructs and stimulated with caspase-3 inhibitor, Z-DEVD-FMK, (20 μ M), 60 min before TNF- α treatment (100 ng/ml) for 72 h. Apoptotic cells were determined by annexin V-FITC/PI method ( a ). After 72 h of treatment with TNF- α , the cells were immunoblotted with specific antibodies for full-length (116 KDa) and cleaved PARP-1 (89 KDa). Tubulin was used as a loading control. Blots shown are representative of three independent experiments ( b–d ). These results were obtained from at least three different experiments, ** P <0.01TNF- α versus TNF- α /Z-DEVD (Mock), * P <0.02 TNF- α versus TNF- α /Z-DEVD (SGK-1 dn); N =3. Results are expressed as means±S.D.

Article Snippet: Z-DEVD-fmk (BD Pharmingen, Milan, Italy): 20 μ M, 60 min before the TNF- α stimulation.

Techniques: Activation Assay, Transfection, Construct