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1.
Fig. 2

Fig. 2. From: Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells.

Over expression of FADD attenuates expression of cFLIP and activates cascades of extrinsic caspases. a Expression of cFLIPL procaspase-8 and caspase-3 in cytosolic fractions was examined by western blot analysis at 24–96 h post transfection of YFP-FADD in HEK 293T cells, empty vector (pEYFP without FADD) transfected HEK 293T cells of 96 h were taken as control. b Activity of caspase 8 in YFP-FADD transfected HEK 293T cells at 24–96 h were determined using IETD-AFC substrate. Empty vector (pEYFP without FADD) transfected cells were taken as control. Error bars represent mean ± SEM from three independent experiments. P value indicates *P ≤ 0.05 and **P ≤ 0.001.

Kishu Ranjan, et al. J Cell Commun Signal. 2012 August;6(3):155-168.
2.
Fig. 3

Fig. 3. From: Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells.

Real-time quantitative PCR analyses for transcriptional expression of cFLIPL, FADD and procaspase-8. a Expression of cFLIPL mRNA at 24–96 h. b Expression of endogenous FADD mRNA at 24–96 h. c Expression of procaspase-8 mRNA at 24–96 h. Total RNA was isolated from YFP-FADD transfected and empty vector (pEYFP without FADD) transfected HEK 293T cells at 24–96 h and after the synthesis of cDNA, RT-qPCR was performed. The values were normalized by using the difference in critical thresholds (CT) between target gene and 18SrRNA (endogenous control). The expression of mRNA of respective genes in FADD transfected HEK 293T cells were compared with control (empty vector transfected HEK 293T cells) using the values of 2−ΔΔCT. Error bars represent mean ± SD from three independent experiments. P value indicates *P ≤ 0.05.

Kishu Ranjan, et al. J Cell Commun Signal. 2012 August;6(3):155-168.
3.
Fig. 6

Fig. 6. From: Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells.

Cell death and expression of cell death regulatory proteins in FADD overexpressed HEK 293T cells upon combined treatment of CD 95L and Cycloheximide (CHX). 48 h post FADD transfected HEK 293T cells were subjected to combine treatment of CD 95L (200 ng/ml) and CHX (5 μg/ml) for 1–4 h. a Cells death analysis by Propidium iodide exclusion assay. Nuclei shown in red display apoptotic cell death. Figures shown are representative of more than 150 cells analyzed from random fields. Scale bar represents 2 μm. b Quantitative measurement of cell death were carried out by trypan blue assay. Control represents 48 h of incubated HEK 293T cells and 48 h post FADD transfected HEK 293T cells without treatment of CHX and CD 95L. Error bars represent mean ± SEM from three independent experiments. c Expression of cFLIPL, and procapsase 8 proteins in cytosolic fraction of 48 h post FADD transfected HEK 293T cells treated with CHX and CD 95L were examined by western blot analyses at given time points. Untreated 48 h post FADD transfected HEK 293T cells were taken as controls and β actin was used as a loading control. d Caspase-8 activity in presence of CD 95L and CHX in individual and in combination on FADD overexpressed HEK 293T cells was determined using IETD-AFC as a substrate. Control represents 48 h of incubated HEK 293T cells and 48 h of FADD transfected HEK 293T cells without treatment of CHX and CD 95L. Error bars represent mean ± SEM from three independent experiments. P value indicates *P ≤ 0.05

Kishu Ranjan, et al. J Cell Commun Signal. 2012 August;6(3):155-168.
4.
Fig. 5

Fig. 5. From: Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells.

Cell death and expression of cell death regulatory proteins in FADD overexpressed HEK 293T cells treated with CHX. a Cycloheximde dependent apoptotic cell death was monitored by Annexin V-FITC/PI staining. HEK 293T cells were subjected to CHX treatment (5 μg/ml) for 2–8 h and stained with Annexin V-FITC and PI. Nuclei shown in red are indicating early induction of cell death. Figures shown are representative of more than 200 cells analyzed of random field. Scale bar represents 2 μm. b Expression of FADD, cFLIPL, and procapsase 8 proteins in the cytosolic fraction of HEK 293T cell stimulated with CHX for 2–8 h was examined by western blot analyses. Untreated HEK 293T cells were taken as an control and β-actin was used as a loading control. c–f 48 h post FADD transfected HEK 293T cells were treated with CHX (5 μg/ml) for 2–8 h and cell death, expression of apoptosis regulatory proteins and activity of caspase 8 were examined. c Propidium iodide staining of 48 h post FADD transfected HEK 293T cells treated with CHX (5 μg/ml) for 2–8 h. Nuclei shown in red display apoptotic cell death. Figures shown are representative of more than 150 cells analyzed from random fields. Scale bar represents 2 μm. d Quantitative measurement of cell death upon treatment of CHX on HEK 293T cells and 48 h post FADD transfected HEK 293T cells was carried out by trypan blue assay. Control represents 48 h of incubated HEK 293T cells and 48 h of FADD transfected HEK 293T cells without treatment of CHX. Error bars represent mean ± SEM from three independent experiments. e Expression of cFLIPL, and procapsase 8 proteins in cytosolic fraction of 48 h post FADD transfected HEK 293T cell stimulated with CHX for 2–8 h was examined by western blot analyses. Without CHX treated 48 h post FADD transfected HEK 293T cells were taken as a control and β-actin was used as a loading control. f Caspase 8 activity was determined in untransfected HEK 293T cells and post 48 h FADD transfected HEK 293T cells upon stimulation with CHX for mentioned time points using IETD-AFC as a substrate. Control represents 48 h of incubated HEK 293T cells and 48 h post FADD transfected HEK 293T cells without treatment of CHX. Error bars represent mean ± SEM from three independent experiments. Error bars represent mean ± SEM from three independent experiments. The P value indicates *P ≤ 0.05

Kishu Ranjan, et al. J Cell Commun Signal. 2012 August;6(3):155-168.
5.
Fig. 1

Fig. 1. From: Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells.

Overexpression of FADD mediated apoptosis in HEK 293T cells: HEK 293T cells were transfected with FADD-pEYFP plasmid. a Cellular localization and morphological analyses of pEYFP-FADD transfected HEK 293T cells at 24–96 h. Left panel is showing empty vector (pEYFP without FADD) transfected HEK 293T cells as a control. Yellow fluorescence is showing expression of FADD in HEK293T cells localize to periphery of cell membrane. Morphology of cells was observed under bright field microscope. Figure shown are representative of more than 150 cells analyzed from random fields, scale bar represents 2 μm. b Localization of YFP-FADD was monitored by DAPI (1 μg/ml) staining on FADD transfected HEK 293T cells at 24–96 h of post transfection. Images showing YFP-FADD and DAPI stained nuclei were co-localized in a common panel. The data shown are representative of more than 150 cells analyzed of random fields. Scale bar represents 2 μm. c Cytosolic expression of YFP-FADD (~ 53 kDa) and endogenous FADD (~27 kDa) was analyzed by western-blot for the designated time points, empty vector (pEYFP without FADD) transfected HEK 293T cells were taken as control and β actin was used as a loading control. d Apoptotic cell death in FADD transfected HEK 293T cells at 24–96 h were examined with propidium iodide staining shown in red observed under fluorescent microscope. YFP-FADD and PI stained nuclei were merged in a common panel. The figure shown is representative of more than 100 cells analyzed of random fields. Scale bar represents 2 μm. e Quantitative measurement of cell death was carried out by trypan blue assay at the 24–96 h. Empty vector (pEYFP without FADD) transfected HEK 293T cells were taken as control. Error bars represent mean ± SEM from three independent experiments. P value indicates *P ≤ 0.05 and **P ≤ 0.001.

Kishu Ranjan, et al. J Cell Commun Signal. 2012 August;6(3):155-168.
6.
Fig. 4

Fig. 4. From: Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells.

Cell death and expression of cell death regulatory proteins in FADD overexpressed HEK 293T cells treated with CD 95L. a Localization of YFP-FADD upon CD 95L treatment (200 ng/ml) was monitored by DAPI (1 μg/ml) counterstaining on 48 h post FADD transfected HEK 293T cells for 1–4 h. Images showing YFP-FADD and DAPI stained nuclei were co-localized in a common panel. Figures shown are representative of more than 150 cells analyzed of random fields. Scale bar represents 2 μm. b Propidium iodide staining for detection of apoptotic cell death upon stimulation of CD 95L (200 ng/ml) on 48 h post FADD transfected HEK 293T cells for 1–4 h. Nuclei shown in red display apoptotic cell death. Figures shown are representative of more than 150 cells analyzed of random fields. Scale bar represents 2 μm. c Quantitative measurement of cell death upon stimulation of CD 95L on HEK 293T cells and 48 h post FADD transfected HEK 293T cells were carried out by trypan blue assay. Control represents 48 h of incubated HEK 293T cells and 48 h of FADD transfected HEK 293T cells without treatment of CD 95L. Error bars represent mean ± SEM from three independent experiments. d Expression of cFLIPL and procapsase 8 proteins in cytosolic fraction of HEK 293T cell stimulated with CD 95L for 1–4 h was examined by western blot analyses. CD 95L untreated cells incubated for 48 h in culture media were taken as a control and β actin was used as a loading control. e Expression of cFLIPL,procapsase 8 and procaspase 3 proteins in cytosolic fraction of 48 h post FADD transfected HEK 293T cell stimulated with CD 95L for 1–4 h was examined by western blot analyses. Without CD 95L treated 48 h post FADD transfected cells were taken as control and β-actin was used as a loading control. f Activity assay of caspase 8 was determined in HEK 293T cells and post 48 h FADD transfected HEK 293T cells upon stimulation with CD 95L for mentioned time points using IETD-AFC as a substrate. Control represents 48 h of incubated HEK 293T cells and 48 h post FADD transfected HEK 293T cells without treatment of CD 95L. Error bars represent mean ± SEM from three independent experiments. Error bars represent mean ± SEM from three independent experiments. The P value indicates *P ≤ 0.05 and **P ≤ 0.001.

Kishu Ranjan, et al. J Cell Commun Signal. 2012 August;6(3):155-168.

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