Journal: Cell death and differentiation

Article Title: Caspases rather than calpains mediate remodelling of the fodrin skeleton during human placental trophoblast fusion.

doi: 10.1038/cdd.2009.133

Figure Lengend Snippet: Figure 4 Effects of calpain inhibitors, general caspase inhibitor or individual caspase inhibitors 3, 8 and 9 on a-fodrin fragmentation in BeWo cells and primary term trophoblasts. (a) Inhibitors of caspases 3, 8 and 9 (20 mM, DEVD, IETD and LEHD, respectively) attenuated the generation of the 120 kDa a-fodrin fragment in forskolin-treated (20 mM) BeWo cells after 48 h cultivation. The inhibitors of calpain – calpeptin and calpain inhibitor III (20 mM, 48 h) – did not affect forskolin-induced fragmentation of a-fodrin in BeWo cells. The general caspase inhibitor (20 mM, Q-VD-OPh) completely blocked a-fodrin fragmentation in forskolin-treated (20 mM, 48 h) BeWo cells. (b) Similar effects were observed in primary term trophoblasts after 48 h cultivation. (c) Efficiency of calpeptin and calpain inhibitor III was tested in ionomycin (10 mM, 2 h)-treated BeWo cells and HUVEC. Although a-fodrin fragmentation was weak and not effected by calpain inhibitors in ionomycin-treated BeWo cells, ionomycin-induced pronounced generation of the 150/145 kDa a-fodrin doublet in HUVEC, which was blocked in the presence of calpeptin and calpain inhibitor III. (d) Forskolin treatment (20 mM, 48 h) led to a slight increase of cleavage products of caspases 3, 8 and 9 (arrows) compared with vehicle control (DMSO, 0.2%). Staurosporine (2 mM, 2 h), but not ionomycin (10 mM, 1.5 h) strongly induced activation of caspases 3, 8 and 9 in BeWo cells. Nitrocellulose membranes were stained with Ponceau S solution to verify equal protein loading and blotting efficiency. Western blots are representative for three independent experiments

Article Snippet: On the next day, cells were preincubated for 30 min with calpeptin (Z-Leu-NLe-CHO, Calbiochem, Darmstadt, Germany), calpain inhibitor III (Z-Val-Phe-CHO, Calbiochem), caspase 3 inhibitor peptide (Z-DEVD-FMK, BD PharMingen, Franklin Lakes, NJ, USA), caspase 8 inhibitor peptide (Z-IETD-FMK, BD PharMingen), caspase 9 inhibitor peptide (Z-LEHD-FMK, R&D Systems, Minneapolis, MN, USA), general caspase inhibitor (Q-VD-OPh, R&D Systems) or negative control peptide (Z-FA-FMK, BD PharMingen) at a concentration of 20mM each.

Techniques: Control, Activation Assay, Staining, Western Blot

Journal: Cell death and differentiation

Article Title: Caspases rather than calpains mediate remodelling of the fodrin skeleton during human placental trophoblast fusion.

doi: 10.1038/cdd.2009.133

Figure Lengend Snippet: Figure 5 Effects of calpain and caspase inhibitors on bhCG secretion and syncytialization in BeWo cells and primary term trophoblasts. (a) Calpeptin and calpain inhibitor III (20 mM), as well as individual caspase inhibitors of caspase 8 (IETD, 20 mM), caspase 3 (DEVD, 20 mM), caspase 9 (LEHD, 20 mM) and the general caspase inhibitor (Q-VD-OPh, 20 mM) decreased forskolin (20 mM)-induced bhCG secretion in BeWo cells after 48 h. Secreted bhCG was normalized to total protein of treated cells. Secretion from BeWo cells treated with forskolin without inhibitor is given as 100%. (b) Inhibitors showed similar effects on bhCG secretion in primary term trophoblasts after 48 h. bhCG secretion from untreated primary term trophoblasts is given as 100%. (c) Syncytialization of forskolin (20 mM)-treated BeWo cells was not affected by calpain inhibitors (calpeptin and calpain inhibitor III, 20 mM) and inhibitors of caspases 3 (DEVD, 20 mM) and 9 (LEHD, 20 mM) after 48 h. Caspase 8 inhibitor (IETD, 20 mM) and the general caspase inhibitor Q-VD-OPh decreased syncytialization to 30.9 and 30.7%, respectively, compared with BeWo cells treated with forskolin alone (36.0 %). Data are presented as mean±S.D. from three independent experiments performed in triplicates. *Pp0.05, **Pp0.01, ***Pp0.001

Article Snippet: On the next day, cells were preincubated for 30 min with calpeptin (Z-Leu-NLe-CHO, Calbiochem, Darmstadt, Germany), calpain inhibitor III (Z-Val-Phe-CHO, Calbiochem), caspase 3 inhibitor peptide (Z-DEVD-FMK, BD PharMingen, Franklin Lakes, NJ, USA), caspase 8 inhibitor peptide (Z-IETD-FMK, BD PharMingen), caspase 9 inhibitor peptide (Z-LEHD-FMK, R&D Systems, Minneapolis, MN, USA), general caspase inhibitor (Q-VD-OPh, R&D Systems) or negative control peptide (Z-FA-FMK, BD PharMingen) at a concentration of 20mM each.

Techniques:

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 1: Expression of p33ING1b and p29ING4 in renal cell carcinoma. (a) mRNA expression of p33ING1b and p29ING4 was increased at early and late stages of disease with higher expression of both genes at late stages of disease compared to early stages (Robson stages III/IV vs. I/II) (p33ING1b and p29ING4: stages I/II and III/IV vs. normal tissue p<0.001 and p<0.0001, respectively; p33ING1b and p29ING4: stages III/IV vs. I/II p<0.01 and p<0.001, respectively). (b) Results of the real time RT-PCR were normalized to kidney normal tissue and expressed as x-fold difference (2- ∆∆Ct). Expression of p33ING1b and p29ING4 by renal carcinoma cells was confirmed (Cy3 red; DAPI blue (nuclear counterstaining)). (c) Sections are representative of n=30 patients/group. Representative western blots of p33ING1b and p29ING4 (loading control ß-actin).

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 4: p33ING1b- and p29ING4-specific T cell reactivity. Four peptides resulted in significantly increased expression of IFN-γ compared to controls, independent of tumor stage (p33ING1b (aa109-118) and aa259-268, and p29ING4 (aa149-158) and aa239-248; p<0.001) Stages I/ II (a) and Stages III/IV (b). Elispot analysis was measured as spots/5 x 105 cells/patient.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Enzyme-linked Immunospot

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 5: p33ING1b- and p29ING4-specific T cell reactivity. Two peptides resulted in increased expression of IL-10 compared to controls; peptide p33ING1b (aa109-118) at all stages and p29ING4 (aa239-248) at late stages (p<0.001). Stages I/ II (a) and Stages III/IV (b). Luminex analysis was measured as pg/ml.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Luminex

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 6: p33ING1b- and p29ING4-specific T cell reactivity. Evaluation of IL-2 expression revealed that only a single epitope (p29ING4 (aa149-158)) resulted in high responsiveness of T cells from RCC patients after stimulation with the peptide (stages I/II vs. III/IV p<0.001). PBMCs from healthy volunteers (n=30) had no significant IL-2 expression after stimulation with all peptide epitopes. Luminex analysis for IL-2 expression was measured as pg/ml and presented as percent stimulation index.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Luminex

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 7: p29ING4 presentation by MHC class I (a) and II (b) molecules. p29ING4 presentation by major histocompatibility complex (MHC) class I and II antigens was shown to be increased in RCC but not in healthy kidneys.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Immunopeptidomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mesenchymal stem cells expressing TRAIL lead to tumour growth inhibition in an experimental lung cancer model

doi: 10.1111/j.1582-4934.2008.00317.x

Figure Lengend Snippet: MSCs transduced with Ad.TR can induce apoptosis in A549 cells. ( A ) MSCs were transduced with Ad.BGal (100 pfu/cell) or Ad.TR (100 pfu/cell). After 48 hrs MSCs were trypsinized and 10 4 of these cells were added to wells that had been seeded the day before with A549 cells at 10 5 cells per well. After 48 hrs, wells were trypsinized and the complete mixed cell population was measured by Nicoletti apoptosis assay. A549 cells and MSCs cultured alone as well as A549 cells mixed with MSCs transduced with Ad.BGal showed only background apoptosis of below 5%. In contrast, in A549 mixed with MSCs transduced with Ad.TR apoptosis rates of almost 30% could be measured. This apoptosis could be inhibited by TRAIL neutralizing antibodies (α-TR-ab) and the pan-caspase inhibitor zVAD. Numbers represent mean values of three samples ± standard deviation. ** P < 0.001. ( B ) MSCs cocultured with A549 cells represent approximately 1% of the final cell population. 10 4 untransduced or Ad.BGal transduced (100 pfu/cell) MSC cells were added to wells that were unseeded or had been seeded the previous day with 10 5 A549 cells. After 48 hrs, cells were detached from wells using trypsin free buffer (Specialty Media, NJ, USA). Cells were stained with anti-CD105 and analysed by flow cytometry. The isotype control for a 1:10 MSC:A549 mix (MSC+A549 ISO) was, as expected, negative for CD105, whereas 98% of all cells in a pure MSC culture (MSC) were CD105 positive. A549 cells only (A549) showed a weak CD105 signal in 0.5% of all cells. In 1:10 mixes with MSCs (MSC+A549) or MSCs transduced with Ad.BGal (A549+MSCβGal) the CD105 signal increased to about 1.5% 48 hrs after mixing and coculturing. Please note that graph is depicted in log-scale. ( C ) The experiment was performed as described above , however, untransduced and transduced (Ad.BGal and Ad.TR) MSC cells were added at 10 4 (1:10) (black bars) and 3.3 × 10 3 (1:30) (white bars) cells per well. After 48 hrs, cells were analysed by Nicoletti apoptosis assay proving that even at a lower ratio against the target cancer cells, MSCs expressing TRAIL can trigger significant levels of apoptosis. Numbers represent mean values of 12 samples ± standard deviation. ** P < 0.001. ( D ) MSCs were transduced with Ad.EGFP (100 pfu/cell) (black bars) or Ad.TR (100 pfu/cell) (white bars). After 48 hrs, MSCs were harvested and 10 4 of these cells were mixed with 10 5 pre-seeded primary human fibroblasts (Fib). After 48 hrs, apoptosis was determined by Nicoletti apoptosis assay. HCT116 cells (HCT) served as positive controls in this experiment.

Article Snippet: The pan-caspase inhibitor zVAD was bought from Santa Cruz (Santa Cruz, CA, USA).

Techniques: Transduction, Apoptosis Assay, Cell Culture, Standard Deviation, Staining, Flow Cytometry, Control, Expressing