Figure 1.
Figure 1. Constitutive NF-κB activation in CTCL cells. (A) CTCL cells constitutively express activated NF-κB. Nuclear extracts from CTCL cell lines (SeAx, HuT-78, MyLa) and from PBLs from 15 patients with Sézary syndrome were analyzed by EMSA. Analysis of SeAx (lane 1), HuT-78 (lane 3), and MyLa (lane 4) CTCL lines, and of PBLs from 15 patients with SS (lanes 5-19), and from one healthy donor as control (lane 20) is presented. N.S. represents a nonspecific unidentified band. Treatment with TNFα (10 ng/mL for 30 minutes) did not increase the constitutive nuclear translocation of NF-κB in SeAx cells (lane 2), and no translocation of NF-κB was detected in nuclear extracts of PBLs from a healthy donor (lane 20). The data shown are representative of 3 independent experiments. (B) Supershift assay of CTCL cell lines. Nuclear extracts from HuT-78, SeAx, and MyLa cells were subjected to supershift assays with specific NF-κB antibodies directed against p50, p65, p52, RelB, and c-Rel subunits. The positions of the retarded NF-κB species are indicated by arrows on the left. Similar results were obtained in 2 independent experiments. (C) Supershift assay of CTCL cells from 3 patients with Sézary syndrome. The assay was performed as described in panel B. (D) Evidence of NF-κB nuclear translocation in CTCL by confocal microscopic analysis. CTCL cells were fixed, permeabilized, and stained with DAPI (nucleus marker, blue); with anti-p65 (1/25); and visualized with anti–rabbit FITC–(green) or Texas Red (red)–labeled secondary Ab. Images were acquired on a Zeiss LSM-510 META laser scanning confocal microscope equipped with a Zeiss Plan Apochromat 63 ×/1.4 NA oil objective, using LSM510 software (version 3.2). Results obtained with CTCL cell lines SeAx and MyLa, PBLs from 2 patients with SS (cases 1 and 20), and from one healthy donor are presented. p50 and p65 proteins are detected in intranuclear and cytoplasmic localizations in CTCL cells only. The proportion of cells showing a positive nuclear staining reached 90% in CTCL lines and 80% in PBLs from SS patients. In contrast, resting PBLs from healthy donors exhibit a cytoplasmic pattern of p50/p65 staining.

Constitutive NF-κB activation in CTCL cells. (A) CTCL cells constitutively express activated NF-κB. Nuclear extracts from CTCL cell lines (SeAx, HuT-78, MyLa) and from PBLs from 15 patients with Sézary syndrome were analyzed by EMSA. Analysis of SeAx (lane 1), HuT-78 (lane 3), and MyLa (lane 4) CTCL lines, and of PBLs from 15 patients with SS (lanes 5-19), and from one healthy donor as control (lane 20) is presented. N.S. represents a nonspecific unidentified band. Treatment with TNFα (10 ng/mL for 30 minutes) did not increase the constitutive nuclear translocation of NF-κB in SeAx cells (lane 2), and no translocation of NF-κB was detected in nuclear extracts of PBLs from a healthy donor (lane 20). The data shown are representative of 3 independent experiments. (B) Supershift assay of CTCL cell lines. Nuclear extracts from HuT-78, SeAx, and MyLa cells were subjected to supershift assays with specific NF-κB antibodies directed against p50, p65, p52, RelB, and c-Rel subunits. The positions of the retarded NF-κB species are indicated by arrows on the left. Similar results were obtained in 2 independent experiments. (C) Supershift assay of CTCL cells from 3 patients with Sézary syndrome. The assay was performed as described in panel B. (D) Evidence of NF-κB nuclear translocation in CTCL by confocal microscopic analysis. CTCL cells were fixed, permeabilized, and stained with DAPI (nucleus marker, blue); with anti-p65 (1/25); and visualized with anti–rabbit FITC–(green) or Texas Red (red)–labeled secondary Ab. Images were acquired on a Zeiss LSM-510 META laser scanning confocal microscope equipped with a Zeiss Plan Apochromat 63 ×/1.4 NA oil objective, using LSM510 software (version 3.2). Results obtained with CTCL cell lines SeAx and MyLa, PBLs from 2 patients with SS (cases 1 and 20), and from one healthy donor are presented. p50 and p65 proteins are detected in intranuclear and cytoplasmic localizations in CTCL cells only. The proportion of cells showing a positive nuclear staining reached 90% in CTCL lines and 80% in PBLs from SS patients. In contrast, resting PBLs from healthy donors exhibit a cytoplasmic pattern of p50/p65 staining.

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