Figure 5
Figure 5. Glucocorticoids induce c-maf degradation through the ubiquitination-proteasome pathway. (A) RPMI 8226 myeloma cells were treated with increasing concentrations of dexamethasone for 48 hours. After treatment, mRNA was extracted. Levels of c-maf and GAPDH were detected by quantitative real-time PCR. Levels of c-maf were normalized for GAPDH expression and expressed as a mean fold change (± SD) over buffer-treated cells. (B) NIH3T3 cells overexpressing c-maf were treated with dexamethasone (DEX; 2 μmol/L) in the absence or presence of the proteasome inhibitor MG132 (MG; 10 μmol/L). After treatment, cell lysates were prepared, normalized for total protein, and analyzed by SDS-PAGE/immunoblotting using antibodies specific for c-maf and β-actin. (C) LP-1 myeloma cells overexpressing c-maf were treated with MG132 (10 μmol/L) and/or dexamethasone (2 μmol/L). After treatment, cell lysates were prepared, normalized for total protein, and analyzed by SDS-PAGE/immunoblotting using antibodies specific for c-maf and b-actin. (D) NIH3T3 cells were cotransfected with c-maf along with HA-ubiquitin (Ub) or GFP cDNA. Twenty-four hours after transfection, cells were lysed. Cell lysates were incubated with anti-HA beads overnight. The resultant precipitates were analyzed by SDS-PAGE/immunoblotting using antibodies specific for c-maf. IP indicates immunoprecipitation; and WB, Western blotting. (E) NIH3T3 cells overexpressing c-maf and the cyclin D2 promoter driving luciferase, and NIH3T3 cells without c-maf but overexpressing the cyclin D2 promoter driving luciferase were treated with dexamethasone (2 μmol/L) and increasing concentrations of MG132. After incubation, cyclin D2 transactivation was measured by luciferase assay and viability measured by MTS assay. The relative luciferase expression represents the mean of 3 independent experiments. (F) LP-1 myeloma cells were treated with increasing concentrations of dexamethasone for 24 hours. After treatment, total mRNA was extracted. Levels of ubiquitin C and GAPDH were measured by quantitative RT-PCR. Ubiquitin C expression was normalized for GAPDH and expressed as a mean fold change (± SD) over buffer-treated cells. (G) NIH3T3 cells overexpressing c-maf were transfected with ubiquitin cDNA or vector control, or treated with dexamethasone (5 μmol/L). Forty-eight hours after transfection, whole-cell lysates were prepared, normalized for total protein, and analyzed by SDS-PAGE/immunoblotting using antibodies specific for c-maf and β-actin. (H) LP-1 cells were transfected with cDNA corresponding to ubiquitin or empty vector. Twenty-four hours after transfection, cell viability was measured by MTS assay and expressed as a mean percentage (± SD; n = 3) relative to control cells.

Glucocorticoids induce c-maf degradation through the ubiquitination-proteasome pathway. (A) RPMI 8226 myeloma cells were treated with increasing concentrations of dexamethasone for 48 hours. After treatment, mRNA was extracted. Levels of c-maf and GAPDH were detected by quantitative real-time PCR. Levels of c-maf were normalized for GAPDH expression and expressed as a mean fold change (± SD) over buffer-treated cells. (B) NIH3T3 cells overexpressing c-maf were treated with dexamethasone (DEX; 2 μmol/L) in the absence or presence of the proteasome inhibitor MG132 (MG; 10 μmol/L). After treatment, cell lysates were prepared, normalized for total protein, and analyzed by SDS-PAGE/immunoblotting using antibodies specific for c-maf and β-actin. (C) LP-1 myeloma cells overexpressing c-maf were treated with MG132 (10 μmol/L) and/or dexamethasone (2 μmol/L). After treatment, cell lysates were prepared, normalized for total protein, and analyzed by SDS-PAGE/immunoblotting using antibodies specific for c-maf and b-actin. (D) NIH3T3 cells were cotransfected with c-maf along with HA-ubiquitin (Ub) or GFP cDNA. Twenty-four hours after transfection, cells were lysed. Cell lysates were incubated with anti-HA beads overnight. The resultant precipitates were analyzed by SDS-PAGE/immunoblotting using antibodies specific for c-maf. IP indicates immunoprecipitation; and WB, Western blotting. (E) NIH3T3 cells overexpressing c-maf and the cyclin D2 promoter driving luciferase, and NIH3T3 cells without c-maf but overexpressing the cyclin D2 promoter driving luciferase were treated with dexamethasone (2 μmol/L) and increasing concentrations of MG132. After incubation, cyclin D2 transactivation was measured by luciferase assay and viability measured by MTS assay. The relative luciferase expression represents the mean of 3 independent experiments. (F) LP-1 myeloma cells were treated with increasing concentrations of dexamethasone for 24 hours. After treatment, total mRNA was extracted. Levels of ubiquitin C and GAPDH were measured by quantitative RT-PCR. Ubiquitin C expression was normalized for GAPDH and expressed as a mean fold change (± SD) over buffer-treated cells. (G) NIH3T3 cells overexpressing c-maf were transfected with ubiquitin cDNA or vector control, or treated with dexamethasone (5 μmol/L). Forty-eight hours after transfection, whole-cell lysates were prepared, normalized for total protein, and analyzed by SDS-PAGE/immunoblotting using antibodies specific for c-maf and β-actin. (H) LP-1 cells were transfected with cDNA corresponding to ubiquitin or empty vector. Twenty-four hours after transfection, cell viability was measured by MTS assay and expressed as a mean percentage (± SD; n = 3) relative to control cells.

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