Figure 6.
Figure 6. CK2 regulates the NF-κB pathway in MM cells. (A) TNF-α-induced IκBα degradation is impaired in MM cells by blockage of CK2. RPMI 8226 cells were incubated for 60 minutes with 0.1% DMSO or with the CK2 inhibitors IQA (25 μM) or K27 (5 μM), and then TNF-α (10 ng/mL) was added to the cultures. At the indicated time points, cytosolic proteins were harvested and 30 μg was used in Western blot analysis with an anti-IκBα-specific antibody (even protein loading was checked to assess α-tubulin levels). Below each Western blot, a graph displays the ratio between the intensity of the bands corresponding to IκBα and α-tubulin, as assessed by densitometric analysis. (B) TNF-α-induced IκBα Ser32 and Ser36 phosphorylation is reduced on CK2 inhibition. RPMI 8226 cells were grown for 60 minutes with 0.1% DMSO (-) or in the presence (+) of the CK2 inhibitor K27 (5 μM) and then stimulated for 0 or 15 minutes with TNF-α (10 ng/mL). Levels of phospho-Ser32 and phospho-Ser36 IκBα were then assessed by immunoblotting 30 μg of proteins with a phosphospecific antibody. (C) IκBα accumulation on CK2 inhibition or down-regulation by RNA interference. Left: RPMI 8226 cells were grown for 12 hours in the presence of medium (-), TNF-α (10 ng/mL), the CK2 inhibitor IQA (25 μM), or both, and then IκBα protein levels were assessed by immunoblotting with an anti-IκBα-specific antibody. Right: Similarly, IκBα levels were determined in MM cells in which CK2 mRNA and protein expression were silenced by siRNAs (even protein loading was checked to assess β-actin levels). (D) Blockage of CK2 reduced NF-κB transcriptional activity in MM cells. RPMI 8226 and OPM2 cells were transiently transfected with an NF-κB-driven luciferase reporter construct and the construct pRL-TK (see “Materials and methods” for details). Twenty-four hours later, the CK2 inhibitor K27 (5 μM) (white bars) or the proteasome inhibitor MG132 (15 μM) () was added to the cultures for 90 minutes, and the cells were stimulated with TNF-α (10 ng/mL) or were left untreated (▪). After 6 hours, total luciferase activity was determined. Values in the graph are shown as percentage of the TNF-α-treated samples. Cell viability was higher than 80% in all the experimental conditions (data not shown). Data represent mean ± SD.

CK2 regulates the NF-κB pathway in MM cells. (A) TNF-α-induced IκBα degradation is impaired in MM cells by blockage of CK2. RPMI 8226 cells were incubated for 60 minutes with 0.1% DMSO or with the CK2 inhibitors IQA (25 μM) or K27 (5 μM), and then TNF-α (10 ng/mL) was added to the cultures. At the indicated time points, cytosolic proteins were harvested and 30 μg was used in Western blot analysis with an anti-IκBα-specific antibody (even protein loading was checked to assess α-tubulin levels). Below each Western blot, a graph displays the ratio between the intensity of the bands corresponding to IκBα and α-tubulin, as assessed by densitometric analysis. (B) TNF-α-induced IκBα Ser32 and Ser36 phosphorylation is reduced on CK2 inhibition. RPMI 8226 cells were grown for 60 minutes with 0.1% DMSO (-) or in the presence (+) of the CK2 inhibitor K27 (5 μM) and then stimulated for 0 or 15 minutes with TNF-α (10 ng/mL). Levels of phospho-Ser32 and phospho-Ser36 IκBα were then assessed by immunoblotting 30 μg of proteins with a phosphospecific antibody. (C) IκBα accumulation on CK2 inhibition or down-regulation by RNA interference. Left: RPMI 8226 cells were grown for 12 hours in the presence of medium (-), TNF-α (10 ng/mL), the CK2 inhibitor IQA (25 μM), or both, and then IκBα protein levels were assessed by immunoblotting with an anti-IκBα-specific antibody. Right: Similarly, IκBα levels were determined in MM cells in which CK2 mRNA and protein expression were silenced by siRNAs (even protein loading was checked to assess β-actin levels). (D) Blockage of CK2 reduced NF-κB transcriptional activity in MM cells. RPMI 8226 and OPM2 cells were transiently transfected with an NF-κB-driven luciferase reporter construct and the construct pRL-TK (see “Materials and methods” for details). Twenty-four hours later, the CK2 inhibitor K27 (5 μM) (white bars) or the proteasome inhibitor MG132 (15 μM) () was added to the cultures for 90 minutes, and the cells were stimulated with TNF-α (10 ng/mL) or were left untreated (▪). After 6 hours, total luciferase activity was determined. Values in the graph are shown as percentage of the TNF-α-treated samples. Cell viability was higher than 80% in all the experimental conditions (data not shown). Data represent mean ± SD.

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