Figure 5
Figure 5. Loss of surface nucleolin and nucleolin-Fas complex sensitizes B-cell lymphomas to Fas-mediated apoptosis. (A) Indicated cells were challenged with the agonistic Fas antibody CH-11 (25 ng/mL) overnight and analyzed for apoptosis levels by Annexin V/7AAD staining and flow cytometry. The nucleolin pKO cells 906S2, 906S5, and 906P1 showed significant increases in sensitivity to agonistic antibody (906S2: P < .001, 906S5: P < .001, 906P1: P < .02). A Fas-sensitive T-cell line, Jurkat, was used as a positive control for Fas activation. Mean value and standard error of the mean (SEM) of 3 or more independent experiments each with 3 replicates are shown. (B) Cells were challenged with FasL (100 ng/mL) overnight and analyzed for apoptosis levels as in (A). The nucleolin pKO cells showed significant increases in FasL sensitivity compared with the nonsilencing control (906S2: P < .001, 906S5: P < .02, 906P1: P < .01). Mean value and SEM of 3 independent experiments each with 3 replicates are shown. (C) Parental BJAB, nonsilencing control, 906P1, and 906S2 cells were subjected to IP of Fas pre-challenge and 1 hour post-challenge with agonistic antibody CH-11 (25 ng/mL). The immunoprecipitated proteins were separated and Fas, caspase-8, and nucleolin were visualized by IB. Note a lack of nucleolin binding to Fas in 906P1 and 906S2 cells. IB of Fas was used as an IP control. Expression levels of nucleolin, Fas, and β-actin in whole-cell extracts as determined by IB analysis as input and loading controls. Representative data from 3 different experiments are shown. (D) Indicated cell lines were incubated with CH-11 (IgM subclass) for 20 minutes and the amount of bound antibody was analyzed by flow cytometry by measuring an anti-IgM–Allophycocyanin secondary antibody signal. The nucleolin pKO cells showed a significant increase in CH-11 signal (906S2: P < .001, 906S5: P < .03, 906P1: P < .04). Mean value and SEM of 3 independent experiments each with 3 replicates are shown. (E) Cells were incubated with FLAG-tagged FasL for 20 minutes and analyzed for the presence of ligand with an anti-FLAG-phycoerythrin secondary antibody by flow cytometry. The nucleolin pKO cells showed significantly increased FasL binding (906S2: P < .01, 906S5: P < .01, 906P1: P < .03). Mean value and SEM of 3 independent experiments each with 3 replicates are shown.

Loss of surface nucleolin and nucleolin-Fas complex sensitizes B-cell lymphomas to Fas-mediated apoptosis. (A) Indicated cells were challenged with the agonistic Fas antibody CH-11 (25 ng/mL) overnight and analyzed for apoptosis levels by Annexin V/7AAD staining and flow cytometry. The nucleolin pKO cells 906S2, 906S5, and 906P1 showed significant increases in sensitivity to agonistic antibody (906S2: P < .001, 906S5: P < .001, 906P1: P < .02). A Fas-sensitive T-cell line, Jurkat, was used as a positive control for Fas activation. Mean value and standard error of the mean (SEM) of 3 or more independent experiments each with 3 replicates are shown. (B) Cells were challenged with FasL (100 ng/mL) overnight and analyzed for apoptosis levels as in (A). The nucleolin pKO cells showed significant increases in FasL sensitivity compared with the nonsilencing control (906S2: P < .001, 906S5: P < .02, 906P1: P < .01). Mean value and SEM of 3 independent experiments each with 3 replicates are shown. (C) Parental BJAB, nonsilencing control, 906P1, and 906S2 cells were subjected to IP of Fas pre-challenge and 1 hour post-challenge with agonistic antibody CH-11 (25 ng/mL). The immunoprecipitated proteins were separated and Fas, caspase-8, and nucleolin were visualized by IB. Note a lack of nucleolin binding to Fas in 906P1 and 906S2 cells. IB of Fas was used as an IP control. Expression levels of nucleolin, Fas, and β-actin in whole-cell extracts as determined by IB analysis as input and loading controls. Representative data from 3 different experiments are shown. (D) Indicated cell lines were incubated with CH-11 (IgM subclass) for 20 minutes and the amount of bound antibody was analyzed by flow cytometry by measuring an anti-IgM–Allophycocyanin secondary antibody signal. The nucleolin pKO cells showed a significant increase in CH-11 signal (906S2: P < .001, 906S5: P < .03, 906P1: P < .04). Mean value and SEM of 3 independent experiments each with 3 replicates are shown. (E) Cells were incubated with FLAG-tagged FasL for 20 minutes and analyzed for the presence of ligand with an anti-FLAG-phycoerythrin secondary antibody by flow cytometry. The nucleolin pKO cells showed significantly increased FasL binding (906S2: P < .01, 906S5: P < .01, 906P1: P < .03). Mean value and SEM of 3 independent experiments each with 3 replicates are shown.

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