Journal: Transplantation

Article Title: Caspase Inhibition During Cold Storage Improves Graft Function and Histology in a Murine Kidney Transplant Model

doi: 10.1097/tp.0000000000002218

Figure Lengend Snippet: FIGURE 1. Effect of the caspase inhibitor Q-VD-OPh on activated caspase-3. Representative blot demonstrating complete inhibition of caspase-3 protein expression by Q-VD-OPh at a concentration of 50 μM, compared with DMSO or saline in mouse RTECs sub- jected to CS/REW. β-actin is used as a protein loading control.

Article Snippet: M-1 (ATCC CRL-2038) RTECs were subjected to CS in cold saline for 24 hours at 4°C and rewarmed (REW) in normal media at 37°C for 24 hours as previously described.16-18 Renal tubular epithelial cells were incubated with either dimethyl sulfoxide (DMSO) or a caspase inhibitor Q-VD-OPh (R&D systems, OPH001) at a final concentration of 50 μM during CS/REW.

Techniques: Inhibition, Expressing, Concentration Assay, Saline, Control

Journal: Transplantation

Article Title: Caspase Inhibition During Cold Storage Improves Graft Function and Histology in a Murine Kidney Transplant Model

doi: 10.1097/tp.0000000000002218

Figure Lengend Snippet: FIGURE 3. Effect of the caspase inhibitor Q-VD-OPh on caspase-3 and caspase-1 activity. A, Q-VD-OPh significantly reduced caspase-3 ac- tivity in mouse RTECs exposed to CS/REW compared with saline or DMSO. (*P < 0.001 vs Q-VD-OPh, *P < 0.01 vs DMSO and #P < 0.001 vs Q-VD-OPh). B, RTECs exposed to CS/REW in presence of saline, DMSO or Q-VD-OPh had no significant change in caspase-1 activity. (P = not significant). Data were analyzed using 1-way ANOVA and post–multiple-comparison Newman-Keuls test. Values are means ± SEM. n = 4 in each group.

Article Snippet: M-1 (ATCC CRL-2038) RTECs were subjected to CS in cold saline for 24 hours at 4°C and rewarmed (REW) in normal media at 37°C for 24 hours as previously described.16-18 Renal tubular epithelial cells were incubated with either dimethyl sulfoxide (DMSO) or a caspase inhibitor Q-VD-OPh (R&D systems, OPH001) at a final concentration of 50 μM during CS/REW.

Techniques: Activity Assay, Saline, Comparison

Journal: Transplantation

Article Title: Caspase Inhibition During Cold Storage Improves Graft Function and Histology in a Murine Kidney Transplant Model

doi: 10.1097/tp.0000000000002218

Figure Lengend Snippet: FIGURE 2. Effect of the caspase inhibitor Q-VD-OPh on tubular cell death. A, Representative flow cytometry images of mouse RTECs ex- posed to CS/REW in presence of saline, DMSO or Q-VD-OPh. B, Cell death percentage was calculated after detection of annexin V–positive and/or PI-positive cells. Cell death was significantly decreased by Q-VD-OPh vs saline or DMSO in RTECs subjected to CS/REW (*P < 0.001 vs Q-VD-OPh; *P < 0.05 vs DMSO and **P < 0.001 vs Q-VD-OPh). Data were analyzed using 1-way ANOVA and post–multiple-comparison Newman-Keuls test. Values are means ± SEM. n = 4 in each group.

Article Snippet: M-1 (ATCC CRL-2038) RTECs were subjected to CS in cold saline for 24 hours at 4°C and rewarmed (REW) in normal media at 37°C for 24 hours as previously described.16-18 Renal tubular epithelial cells were incubated with either dimethyl sulfoxide (DMSO) or a caspase inhibitor Q-VD-OPh (R&D systems, OPH001) at a final concentration of 50 μM during CS/REW.

Techniques: Flow Cytometry, Saline, Comparison

Journal: Carcinogenesis

Article Title: Smad7 induces plasticity in tumor-infiltrating Th17 cells and enables TNF-alpha-mediated killing of colorectal cancer cells.

doi: 10.1093/carcin/bgu027

Figure Lengend Snippet: Fig. 5. (A) MC38 cells were stimulated with medium or 1:100 diluted supernatants of ex vivo activated TILs isolated from tumors developed in adoptively transferred Rag1−/− mice. After 12 h, MC38 cells were stained with AV and PI and analyzed by flow cytometry. Representative density plots are shown from three independent experiments. Numbers in the quadrants indicate the frequency of cell subpopulations. (B) AV/PI staining was evaluated in MC38 stimulated as in A at different time points as indicated. (C) AV/PI staining of MC38 cells after 24 h stimulation with supernatants of activated TILs isolated from the different groups of mice in the presence of IL17A, TNF-α and IFN-γ neutralizing antibodies as indicated. (D) AV/PI staining of MC38 cells after 24 h stimulation with IL17A, TNF-α and IFN-γ as indicated. (E) Caspase 8 staining of MC38 cells after 6 h stimulation with IL17A, TNF-α and IFN-γ as indicated. (F) AV/PI staining of MC38 cells after 24 h stimulation with TNF-α, IFN-γ or Caspase inhibitors as indicated. (G) Caspase 8 staining of MC38 cells stimulated with medium or supernatants obtained from activated TILs isolated from the different groups of adoptively transferred Rag1−/− mice. Representative density plots are shown and numbers above the gates represent the frequency of Caspase 8-positive cells. (H) AV/PI staining of MC38 cells stimulated with medium or supernatants of 24 h-activated TILs from the different groups of adoptively transferred Rag1−/− mice in the presence or absence of the pan-caspase (Q-VD-OPH) inhibitor as indicated. Representative density plots are shown from three independent experiments. Numbers in the quadrants indicate the frequency of cell subpopulations. Bars in B, C, D and F indicate the average of AV/PI-positive MC38 cells and E indicates the average of Caspase 8-positive MC38 cells (±SD) obtained from three independent experiments.

Article Snippet: In some experiments, general caspase inhibitor (Q-VD-OPH, 100 μM; R&D Systems) was used as indicated.

Techniques: Ex Vivo, Isolation, Staining, Flow Cytometry

Journal: Animal Cells and Systems

Article Title: The effect of caspase-3 inhibition on interdigital tissue regression in explant cultures of developing mouse limbs

doi: 10.1080/19768354.2012.678386

Figure Lengend Snippet: Figure 2. Caspase-3 correlation with apoptosis and proliferation in the mouse front limbs in vivo (AL) and quantification of active caspase-3 positive cells (MO). Collocalization of caspase-3 immunohistochemistry (IHC) (A, D, G, and J), TUNEL assay (B, E, H, and K), and PCNA immunohistochemistry (C, F, I, and L) in serial sections at E12.5 (AC), at E13.5 (DF), at 14.5 (GI), and at 15.5 (JL). Positive cells are brown (black arrows), negative blue (white arrows), magnification 200. Flow cytometry of interdigital (M) and digital (N) cells at 13.5 and percentage comparisons after multiple measurements (O).

Article Snippet: Penetration of the inhibitor in the culture was confirmed using a biotinylated pan caspase inhibitor (R&D System, FMK 011) (data not shown).

Techniques: In Vivo, Immunohistochemistry, TUNEL Assay, Flow Cytometry

Journal: Animal Cells and Systems

Article Title: The effect of caspase-3 inhibition on interdigital tissue regression in explant cultures of developing mouse limbs

doi: 10.1080/19768354.2012.678386

Figure Lengend Snippet: Figure 3. Pharmacological inhibition of caspase-3 in mouse limb explant cultures. (AC) control group, (DF) inhibited group, magnification 200. (A and D) Caspase-3 activation in serial sections after 24 hours of cultivation based on IHC shows no active caspase-3 in the inhibited samples. (B and E) TUNEL assay in serial sections after 24 hours of cultivation shows dramatic reduction of apoptosis in the inhibited samples. (C and F) Comparable growth of the explants cultures checked using PCNA immunohistochemistry (IHC). (G) Macroscopic view on the cultivation starting point stage limb. (H) Control limb after 48 hours of cultivation with loss of the interdigit tissue. (I) Caspase-3-inhibited explant showing interdigital webbing. (J) Caspase-3 was negative after 72 h of cultivation but (K) TUNEL positive cells reappeared and (L) digit separation continued.

Article Snippet: Penetration of the inhibitor in the culture was confirmed using a biotinylated pan caspase inhibitor (R&D System, FMK 011) (data not shown).

Techniques: Inhibition, Control, Activation Assay, TUNEL Assay, Immunohistochemistry

Journal: Molecules

Article Title: Synthesis and Pro-Apoptotic Effects of Nitrovinylanthracenes and Related Compounds in Chronic Lymphocytic Leukaemia (CLL) and Burkitt’s Lymphoma (BL)

doi: 10.3390/molecules28248095

Figure Lengend Snippet: Inhibitor studies: Compound 11h in HG-3 and PGA-1 CLL cell lines—Annexin V/PI FACS analysis (48 h). The HG-3 and PGA-1 CLL cells (5 × 10 4 cells/mL) were pre-treated at 37 °C with either 5 mM N -acetylcysteine (NAC) for 1 h or 40 μM caspase inhibitor C (Z-VAD-FMK) for 4 h prior to 5 μM compound treatment for 48 h.

Article Snippet: The HG-3 and PGA-1 CLL cells (5 × 10 4 cells/mL) were pre-treated at 37 °C with either 5 mM N-acetylcysteine (NAC) for 1 h or 40 μM caspase inhibitor (Z-VAD-FMK; MBL International, Co., Woburn, MA, USA) for 4 h prior to treatment with (11 h at 5 μM) for 48 h. Annexin V/PI FACS analysis was then carried out as described above in .

Techniques:

Journal: Journal of Translational Medicine

Article Title: Specifically targeting ERK1 or ERK2 kills Melanoma cells

doi: 10.1186/1479-5876-10-15

Figure Lengend Snippet: ERK1 and/or ERK2 shRNA triggers caspase cascade including PARP cleavage, and caspase inhibitor partial block of killing of A375 cells . A . Western blot analysis of caspase 3, caspase 8 and PARP including both intact (inactive) and cleaved (active) forms in A375 cells on days 2, 4 and 6 following exposure to shRNAs. GAPDH confirms equivalent loading. B) . Addition of pan-caspase inhibitor, ZVAD (20 mM; last 2 days) reduces killing of A375 cells triggered by ERK1 and/or ERK2 shRNA. Histograms represent the means +/- SEM from 3 independent experiments. Statistical significance portrayed as follows: * p < 0.01, ** p < 0.02, *** p < 0.002.

Article Snippet: Pan-caspase inhibitor ZVAD was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: shRNA, Blocking Assay, Western Blot