Figure 4
Figure 4. Sp1 is the key suppressor of LMP1-hampered EPHA4 promoter activity. (A) LCLs were infected with an shSp1-expressing lentivirus for 5 days and the infected cells were selected with 2 µg/mL puromycin for 2 days. EphA4, Sp1, and β-actin expression levels were detected by western blotting. EphA4 protein-relative folds were normalized to β-actin and standardized with vector control shLuc. (B) BJAB cells were infected simultaneously with LMP1 and shSp1-expressing lentiviruses for 3 days and infected cells were selected with 2 µg/mL puromycin for 2 days. EphA4, Sp1, LMP1, and β-actin were determined by western blotting. EphA4 protein-relative folds were normalized to β-actin and standardized with pSIN plus shLuc controls. (C) LCLs were coinfected with shSp1 and GFP-tagged EPHA4 promoter (−1000 ∼ +42)-expressing lentiviruses for 3 days and then the infected cells were selected with 2 µg/mL puromycin for 2 days. EphA4-relative luciferase activity was first normalized to GFP, followed by standardization with the vector pCDHGL3 (***P < .001, Student t test). Sp1, LMP1, and internal control β-actin were analyzed by western blotting (bottom panel). (D) TW01 cells were infected with an shSp1-expressing lentivirus for 3 days and the infected cells were selected with 2 µg/mL puromycin for 2 days. The Sp1-knockdown TW01 cells were cotransfected with LMP1 plasmid or its vector control pSG5, combined with reporter plasmids EPHA4 promoter (−1000 ∼ +42) or vector control pGL3 and internal control pEGFPC1 plasmids for 2 days. EphA4-relative luciferase activity was first normalized to GFP, followed by standardization with the control vector pGL3 (**P < .01, Student t test). Expression of Sp1, LMP1, and β-actin proteins was analyzed by western blotting (bottom panel). (E) TW01 cells were cotransfected with LMP1 or pSG5 plasmids, reporter plasmids of pGL3 vector control or EPHA4 promoter (−1000 ∼ +42) and internal control pEGFPC1 plasmids. Twenty-four hours posttransfection, 500 nM mithramycin was added to the cells for another 24 hours. EphA4-relative luciferase activity was determined as described previously (*P < .05, Student t test). Expression levels of LMP1 and β-actin were measured by western blotting (bottom panel). (F) BJAB cells were transduced with pSIN- or LMP1-expressing lentiviruses for 5 days and a chromatin immunoprecipitation assay was performed as described previously. DNA-protein complexes were immunoprecipitated using anti-Sp1 Ab or isotype control rabbit immunoglobulin G (IgG). EPHA4 promoter and control GAPDH promoter DNA were detected in the immunoprecipitates by PCR. Total DNA was harvested from BJAB cells and used as the input control.

Sp1 is the key suppressor of LMP1-hampered EPHA4 promoter activity. (A) LCLs were infected with an shSp1-expressing lentivirus for 5 days and the infected cells were selected with 2 µg/mL puromycin for 2 days. EphA4, Sp1, and β-actin expression levels were detected by western blotting. EphA4 protein-relative folds were normalized to β-actin and standardized with vector control shLuc. (B) BJAB cells were infected simultaneously with LMP1 and shSp1-expressing lentiviruses for 3 days and infected cells were selected with 2 µg/mL puromycin for 2 days. EphA4, Sp1, LMP1, and β-actin were determined by western blotting. EphA4 protein-relative folds were normalized to β-actin and standardized with pSIN plus shLuc controls. (C) LCLs were coinfected with shSp1 and GFP-tagged EPHA4 promoter (−1000 ∼ +42)-expressing lentiviruses for 3 days and then the infected cells were selected with 2 µg/mL puromycin for 2 days. EphA4-relative luciferase activity was first normalized to GFP, followed by standardization with the vector pCDHGL3 (***P < .001, Student t test). Sp1, LMP1, and internal control β-actin were analyzed by western blotting (bottom panel). (D) TW01 cells were infected with an shSp1-expressing lentivirus for 3 days and the infected cells were selected with 2 µg/mL puromycin for 2 days. The Sp1-knockdown TW01 cells were cotransfected with LMP1 plasmid or its vector control pSG5, combined with reporter plasmids EPHA4 promoter (−1000 ∼ +42) or vector control pGL3 and internal control pEGFPC1 plasmids for 2 days. EphA4-relative luciferase activity was first normalized to GFP, followed by standardization with the control vector pGL3 (**P < .01, Student t test). Expression of Sp1, LMP1, and β-actin proteins was analyzed by western blotting (bottom panel). (E) TW01 cells were cotransfected with LMP1 or pSG5 plasmids, reporter plasmids of pGL3 vector control or EPHA4 promoter (−1000 ∼ +42) and internal control pEGFPC1 plasmids. Twenty-four hours posttransfection, 500 nM mithramycin was added to the cells for another 24 hours. EphA4-relative luciferase activity was determined as described previously (*P < .05, Student t test). Expression levels of LMP1 and β-actin were measured by western blotting (bottom panel). (F) BJAB cells were transduced with pSIN- or LMP1-expressing lentiviruses for 5 days and a chromatin immunoprecipitation assay was performed as described previously. DNA-protein complexes were immunoprecipitated using anti-Sp1 Ab or isotype control rabbit immunoglobulin G (IgG). EPHA4 promoter and control GAPDH promoter DNA were detected in the immunoprecipitates by PCR. Total DNA was harvested from BJAB cells and used as the input control.

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