Journal:

Article Title: CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway

doi: 10.1093/emboj/19.19.5123

Figure Lengend Snippet: Fig. 4. CD95 expression, susceptibility to apoptosis and CD95/ezrin co-localization in activated human primary T lymphocytes. (A) CD95 membrane expression and apoptosis were analyzed in electronically gated CD4+ by three-color immunofluorescence and FACS analysis in day 1- (upper panels) and day 6- (lower panels) activated human primary lymphocytes (PBL). Left panels are the CD95+/CD69+ CD4+ PBL; central panels are the CD95+/CD45RO+ CD4+ lymphocytes; right panels are the apoptotic cells (FITC–annexin V+) CD4+ PBL after triggering with an anti-CD95 mAb (see Materials and methods). The results are representative of four experiments. (B) CD95 (green, FITC)/ezrin (red, TRITC) localization in day 1- (left panel) and day 6- (right panel) activated human primary T lymphocytes. In the upper and lower panels the single and double stainings are shown, respectively. Note the co-localization in day 6-activated lymphocytes, as revealed by the yellow staining (IVM) (magnification ×1000).

Article Snippet: For cytofluorimetric evaluation of apoptosis of untreated and CD95, triggered CEM cells (125 ng/ml of IgM anti-CD95 antibody) or PBMC (500 ng/ml of IgM anti-CD95 antibody) were washed and double stained by using annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (Eppendorf, Milan, Italy).

Techniques: Expressing, Immunofluorescence, Staining

Journal:

Article Title: CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway

doi: 10.1093/emboj/19.19.5123

Figure Lengend Snippet: Fig. 5. Cytochalasin D effects on CEM cell morphology, CD95 distribution and CD95-mediated apoptosis. SEM analysis of cytochalasin D (CD)-treated (A) and untreated (B) CEM cells. Note the disappearance of uropoidal shape with preservation of microvillar structures, small pseudopodia and ruffle spreading in CD-treated cells (magnification ×35 000). (C) Immunofluorescence analysis of CD95 distribution on CEM cells following cytochalasin D (CD) pre-treatment. Note the scattered distribution of CD95 protein throughout the cell cytoplasm (magnification ×1000). (D) Effects of CD pre-treatment on CD95-mediated apoptosis compared with the apoptosis induced by UVB, or staurosporin. Histograms represent mean ± SD of five different experiments. *Student’s t-test, P <0.001.

Article Snippet: For cytofluorimetric evaluation of apoptosis of untreated and CD95, triggered CEM cells (125 ng/ml of IgM anti-CD95 antibody) or PBMC (500 ng/ml of IgM anti-CD95 antibody) were washed and double stained by using annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (Eppendorf, Milan, Italy).

Techniques: Preserving, Immunofluorescence

Journal:

Article Title: CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway

doi: 10.1093/emboj/19.19.5123

Figure Lengend Snippet: Fig. 7. Effects of phosphorothioate oligonucleotides (PONs) on CD95-mediated apoptosis. (A) Western blotting with mAbs to human ezrin and moesin in CEM cells lysates following sense or antisense PONs treatment or without treatment. Actin levels in the relative lysates are shown. (B) Flow cytometric analysis of CEM cells after double staining procedure with annexin V–FITC/PI performed on living cells. As specified in the control panel (upper left), in the upper right quadrants (annexin V–FITC/PI positive) and in the lower right quadrants (annexin V single positive) of all the panels are represented the cells in the early or late apoptosis, respectively. CEM cells were left untreated (upper left), treated with the anti-CD95 triggering mAb (upper right), treated with the anti-CD95 triggering mAb after pre-treatment with ezrin PONs (central panels) or moesin PONs (lower panels). SEM (left panel) and CD95 distribution, as detected by immunofluorescence (central panel) and immunocytochemistry (right panel), in CEM cells treated with ezrin antisense (C) or sense (D) PONs.

Article Snippet: For cytofluorimetric evaluation of apoptosis of untreated and CD95, triggered CEM cells (125 ng/ml of IgM anti-CD95 antibody) or PBMC (500 ng/ml of IgM anti-CD95 antibody) were washed and double stained by using annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (Eppendorf, Milan, Italy).

Techniques: Western Blot, Double Staining, Immunofluorescence, Immunocytochemistry