Figure 3.
Figure 3. PML regulates c-Jun transcriptional activation upon UV irradiation. (A) PML coactivates c-Jun upon UV irradiation. Activation of GAL4–c-Jun by PML in MEFs. Pml+/+ MEFs were transfected with the GAL4-Luciferase reporter construct and the indicated combinations of GAL4–c-Jun and PML. After 36 hours, cells were left untreated (▦) or UV-irradiated with 40 J/m2 (▪). Reporter activity was measured 3 hours after irradiation. Luciferase is expressed as arbitrary light units and normalized to an internal Renilla luciferase control. Data shown are the mean ± SD of 3 independent experiments performed in triplicate. (B) PML is required for UV-dependent activation of GAL4–c-Jun. Pml+/+ (▦) and Pml-/- (▪) MEFs were transfected with the GAL4-Luciferase reporter construct and the indicated combinations of GAL4–c-Jun and PML and subsequently UV-treated (40 J/m2) for 3 hours. Data shown are the mean ± SD of 3 independent experiments performed in triplicate. (C) The transcriptional activity of endogenous c-Jun is impaired in Pml-/- cells. Pml+/+ and Pml-/- MEFs were transfected with a jun2CRE-Luc reporter plasmid. After 24 hours, transfected cells were left untreated (▦) or UV-treated with 60 J/m2 (▪). Reporter activity was measured 3 hours after irradiation and normalized to an internal Renilla luciferase control. UV-triggered jun2CRE-Luc transcription is expressed as fold induction over luciferase activity in untreated cells. Luciferase activity in untreated cells is arbitrarily shown as 1.0. Data shown are the mean ± SD of 3 independent experiments performed in duplicate. (D) In vivo c-Jun binding to CRE sites is induced by UV irradiation and is defective in the absence of PML. Pml+/+ and Pml-/- MEFs (indicated as +/+ and-/-) were left untreated or UV-irradiated (40 J/m2) and then cultured for 2 hours. Cross-linked chromatin from untreated or UV-treated Pml+/+ and Pml-/- cells was incubated with anti–c-Jun antibody or with control rabbit immunoglobulin G (IgG). Immunoprecipitates were analyzed by PCR with primers specific for jun1 and jun2 regions of the c-jun promoter and for the collagenase (coll) promoter. A sample representing 0.5% of total input chromatin (input) was included in the PCR reactions. Data shown are representative of 3 independent experiments.

PML regulates c-Jun transcriptional activation upon UV irradiation. (A) PML coactivates c-Jun upon UV irradiation. Activation of GAL4–c-Jun by PML in MEFs. Pml+/+ MEFs were transfected with the GAL4-Luciferase reporter construct and the indicated combinations of GAL4–c-Jun and PML. After 36 hours, cells were left untreated (▦) or UV-irradiated with 40 J/m2 (▪). Reporter activity was measured 3 hours after irradiation. Luciferase is expressed as arbitrary light units and normalized to an internal Renilla luciferase control. Data shown are the mean ± SD of 3 independent experiments performed in triplicate. (B) PML is required for UV-dependent activation of GAL4–c-Jun. Pml+/+ (▦) and Pml-/- (▪) MEFs were transfected with the GAL4-Luciferase reporter construct and the indicated combinations of GAL4–c-Jun and PML and subsequently UV-treated (40 J/m2) for 3 hours. Data shown are the mean ± SD of 3 independent experiments performed in triplicate. (C) The transcriptional activity of endogenous c-Jun is impaired in Pml-/- cells. Pml+/+ and Pml-/- MEFs were transfected with a jun2CRE-Luc reporter plasmid. After 24 hours, transfected cells were left untreated (▦) or UV-treated with 60 J/m2 (▪). Reporter activity was measured 3 hours after irradiation and normalized to an internal Renilla luciferase control. UV-triggered jun2CRE-Luc transcription is expressed as fold induction over luciferase activity in untreated cells. Luciferase activity in untreated cells is arbitrarily shown as 1.0. Data shown are the mean ± SD of 3 independent experiments performed in duplicate. (D) In vivo c-Jun binding to CRE sites is induced by UV irradiation and is defective in the absence of PML. Pml+/+ and Pml-/- MEFs (indicated as +/+ and-/-) were left untreated or UV-irradiated (40 J/m2) and then cultured for 2 hours. Cross-linked chromatin from untreated or UV-treated Pml+/+ and Pml-/- cells was incubated with anti–c-Jun antibody or with control rabbit immunoglobulin G (IgG). Immunoprecipitates were analyzed by PCR with primers specific for jun1 and jun2 regions of the c-jun promoter and for the collagenase (coll) promoter. A sample representing 0.5% of total input chromatin (input) was included in the PCR reactions. Data shown are representative of 3 independent experiments.

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