PMC

HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection in the absence or presence of an HTLV-I infectious molecular clone (pACH; 800 ng) and either a control (EGFP), Foxp3, or ΔFKH expression vector (800 ng). Expression of HTLV-I Tax from the molecular clone was measured by quantitative RT-PCR (TaqMan).

(A and B) HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection with NF-κB (A) or HTLV-I LTR (B) luciferase reporter vectors (200 ng) in the presence or absence of an HTLV-I Tax, Foxp3, ΔFKH, or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity was normalized to the internal reference control.
(C) Expression of HTLV-I Tax in (A) and (B) was measured by quantitative RT-PCR (TaqMan) to demonstrate that Foxp3 did not suppress Tax transactivation of NF-κB and the HTLV-I LTR by down-regulating the expression of Tax.
(D) HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection with a Gal4 luciferase reporter vector (200 ng) in the presence or absence of a Gal4-BD or Gal4-BD-Tax expression vector (500 ng), Foxp3, ΔFKH, or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity was normalized to the internal reference control.

(A and B) HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection with an NF-κB luciferase reporter vector (200 ng) in the presence or absence of indicated concentrations of a Foxp3 expression vector or a control vector and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity (A) and Foxp3 mRNA expression (B).
(C) HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection with an NF-κB luciferase reporter vector (200 ng) in the presence or absence of a Foxp3 expression vector or a control vector (1,500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24, 48, 72, and 96 h posttransfection and analyzed for luciferase activity.
(D) CD4⁺ T cells (2 × 10⁶) from three healthy donors were nucleofected with an NF-κB luciferase reporter vector (1,000 ng) and Foxp3 or control expression vectors (2,000 ng) and internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity shown in A, C, and D was normalized to the internal reference control.

(A) HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection with a CREB or an HTLV-I LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
(B) Purified CD4⁺ T cells (2 × 10⁶) from two healthy donors were transfected with a CREB luciferase reporter vector (2,000 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (2,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 1,000 ng).
(C) Purified CD4⁺ T cells (2 × 10⁶) from healthy donors were transfected with an HTLV-I LTR luciferase reporter vector (2,000 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (2,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 1,000 ng).
(D) HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection with a Gal4 luciferase reporter vector (200 ng) in the presence or absence of Gal4-BD, Gal4-BD-CREB-1, or Gal4-BD-c-Jun (500 ng) along with Foxp3 or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells from A, B, C, and D were harvested at 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity was normalized to the internal reference control.
(E) Foxp3, ΔFKH, and CREB-1 expression in whole-cell extracts (20 μg) derived from HEK 293T cells transfected with a control expression vector (EGFP), Foxp3, or ΔFKH expression vectors were analyzed by Western blot analysis. β-actin expression was analyzed as a loading control.

(A and B) HEK 293T cells (5 × 10⁵) (A) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 10⁶) (B) were seeded into six-well plates on the day of transfection with an HIV-1 LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.
(C) Schematic representation of HIV-1 LTR luciferase reporter constructs pHIV-1 LTR-luc and pHIV-1 Δ-κB LTR-luc.
(D and E) CD4⁺ T cells (2 × 10⁶) from two healthy donors were nucleofected with either parental HIV-1 LTR (D) or HIV-1 LTR lacking NF-κB binding sites (at −102 to −81) (E) (1,000 ng), Foxp3 or control expression vectors (2,000 ng), and an internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity shown in A, B, D, and E was normalized to the internal reference control.

(A and B) HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection with a CREB or an HTLV-I LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, DFKH, or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Forskolin (10 μM) was added to the appropriate reactions 20 h posttransfection. Reactions were assayed 24 h posttransfection for luciferase activity (A) and CREB-1 and ATF-2 DNA-binding activity (B).
(C) HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection with a Gal4 luciferase reporter vector (200 ng) in the presence or absence of Gal4-BD-CREB-1 (500 ng) along with a control, Foxp3, or p300 expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity in (A and C) was normalized to the internal reference control.
(D) Whole-cell extracts from HEK 293T cells transfected with a control (EGFP) or Foxp3 expression vector together with a p300-HA expression vector were immunoprecipitated with anti-HA monoclonal antibody. Proteins were then separated by SDS-PAGE and immunoblotted with anti-Foxp3 (ab10563) to detect immunoprecipitates and with anti-HA for the lysates.

(A) Schematic representation of human Foxp3, including proline-rich (P-P-P), zinc finger (ZNF), leucine zipper (ZIP), and forkhead (FKH) domains. Mutations within the Foxp3 gene associated with IPEX have been previously described [,,]. The FKH domain contains a nuclear localization signal (NLS), which is required for Foxp3 expression in the nucleus.
(B and C) HEK 293T cells (5 × 10⁵) (B) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 10⁶) (C) were seeded into six-well plates on the day of transfection with an NF-κB luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity was normalized to the internal reference control.
(D) CD4⁺ T cells (2 × 10⁶) from healthy donors were nucleofected with an NF-κB reporter plasmid (2,000 ng), together with either a Foxp3, ΔFKH, or control expression vector (2,000 ng), and an internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity was normalized to the internal reference control. Results from one healthy donor are shown.
(E) Foxp3, ΔFKH, and NF-κB p65 expression in whole-cell extracts (20 μg) derived from HEK 293T cells transfected with a control expression vector (EGFP), Foxp3, or ΔFKH expression vectors were analyzed by Western blot analysis. β-actin expression was analyzed as a loading control.