A. Electro mobility shift assay for NF-κB DNA binding activity. HEKa and the UM-SCC cell lines indicated in the figure were cultured as described in methods. 5 μg of nuclear extract protein from each cell line was used for binding to radiolabeled oligonucleotide containing the NF-κB-response element. A variable increase in NF-κB-response element DNA binding activity was detected in most UM-SCC cells, when compared to HEKa cells. Free probe was used as negative control. Wild type and mutant cold oligonucleotide were incubated with nuclear extracts from UM-SCC-5 as an NF-κB response element specific binding control. Oct as a control for nuclear extract integrity and loading is shown in overlay. B. NF-κB Reporter Activity from UM-SCC Cells. NF-κB reporter activity was measured by co-transfecting cells with pNF-κB-cis-luciferase reporter plasmid containing a 5x NF-κB enhancer consensus element. NF-κB reporter activities that are presented here represent an average of multiple measurements that were normalized as a percentage of activity of UM-SCC-6 cells. Cell lines shown here include UM-SCC1, 6, 9, 11A, and 11B as indicated. The transfection conditions were optimized to reach over 90% transfection efficiency for each cell line as determined by β-Galactosidase gene under control of a constitutive CMV promoter (bottom panel). Variable NF- κB reporter activities were observed. UM-SCC-6 cells showed significantly high NF-κB reporter activity comparing to other cell lines (p<0.01) while UM-SCC-9 cells showed the significantly low NF-κB reporter activity comparing to other cells (p<0.01). C. IκBα expression and Degradation Time Course From UM-SCC-6 and -9Cells Following Cyclohexmide Treatment. Cells were treated with 50μg/ml cycloheximide for 0.5 to 4 hours as indicated. The protein levels of IκBα were detected by immunoblot analysis and normalized to total protein detected from the membrane. • ♦ UM-SCC-6, UM-SCC-9 cells. Data show that the IκBα protein level in UM-SCC-6 cells is 1.8 fold of that in UM-SCC-9 cells and that the half life of IκBα in UM-SCC-6 cells is 1.5 hr, half of that in UM-SCC-9 cells (3 hours). D. Comparison of IKK Kinase Activity Between UM-SCC-6 And -9 Cells. Cell lysates from UM-SCC-6 and −9 cells were immunoprecipitated by NEMO. IKK kinase activity was measured as incorporated ^[33]P-IκBα by using an N-terminal IκBα-GST fusion protein.as substrate (WT). The result is normalized by total IκBα and, then, IKK2 protein detected from the same membrane. A mutant S32G/S36A N-terminal IκBα-GST fusion protein was used as negative control (Mut). Data show that IKK kinase activity in UM-SCC-6 (SCC-6) cells is one fold higher than that in UM- SCC-9 (SCC-9) cells. The detected gel bands and corresponding measurements of the intensities of these bands are shown under the chart and in the table respectively.

A. Apigenin inhibits Aberrant NF-κB reporter activity from UM-SCC cells. UM-SCC-6, 9, 11A and 11B cells, as indicated in the figure, were exposed to 100 μM Apigenin for 2 hours prior to the DNA transfection for NF-κB luciferase reporter assay. The reporter activities from each cell lines were normalized to their corresponding untreated-control cells (open column, Control; solid column, Apigenin). The data showed that Apigenin inhibited ~90% of the NF-κB reporter activity from UM-SCC-6 cells (p<0.005) and 70% of that from UM-SCC-9 cells (p<0.05). 90% and 80% reduction of NF-κB reporter activities were also observed from UM-SCC-11A (p<0.005) and UM-SCC-11B cells (p<0.05). B and C, CK2β siRNAs knocks down mRNA levels in UM-SCC-6 and UM-SCC-9 cells. UM-SCC-6 and -9 cells were transfected with CSNK2B (CK2β), scrambled, or a universal control siRNAs for 24, 48 and 72 hours as indicated. Untreated, no siRNA transfection; Ctrl-Universal, Universal negative control siRNA from Ambion; Ctrl-Scramble, Stealth scrambled siRNA control for CK2β siRNA; CSNK2B, siRNA specially targeting CK2β mRNA. Data show a }time dependent decrease of CK2β mRNA levels from both cell lines (UM-SCC-6, p<10⁻⁹; UM-SCC-9 cells, p<10⁻⁵). D and E. CK2β siRNA inhibits the Aberrant NF-κB activity in UM-SCC cells. Cells were transfected with siRNAs targeting CK2β subunits as well as control siRNAs, as indicated, for 48 hours prior to the transfection for NF-κB luciferase reporter assay. D. The NF-κB reporter activities from UM-SCC-6 cells; E. The NF-κB reporter activities from UM-SCC-9 cells. CK2β siRNA significantly reduces the NF-κB reporter activity (90%) from UM-SCC-6 cells (p<0.05). ~60% reduction of the NF-κB reporter activity was observed from UM-SCC-9 cells, but this change is not statistically significant. F. CK2β siRNA inhibits expression of endogenous NF-κB inducible gene IκBα and NFKB2/p100 in UM-SCC cells. Messenger RNA was isolated from cell lysates of untreated UM-SCC-6 cells (open bar) and cells transfected with siRNAs targeting CK2β subunits (slashed bar) as well as the scramble control siRNA (solid bar) after 48 hours. A Quantigene kit was used for mRNA quantification of IκBα and NFKN2/p100 as indicated. Data were normalized to its corresponding 18SrRNA level as detected at the same time. Data show statistically significant decrease of IκBα (p<10⁻⁶) and NFKB2/p100 (p<10⁻⁷) in CK2β siRNA transfected cells compared to the corresponding scramble control.

A. IKK2 Is a Substrate of CK2. rIKK2 was incubated without (−) or with 0.5 (0.5) and 5ng (5) of rCK2α and 5ng of rCK2α along with 40μM apigenin (5+Apigenin), as indicated, in the presence of ^[33]P-GTP followed by SDS-PAGE gel separation, transfer, autoradiography and immunoblot analysis for detection of phosphorylated IKK2 and protein levels of IKK2 and CK2α. CK2 phosphorylation of IKK2 activity is presented as the radiation activity detected by autoradiography and normalized to the protein levels of IKK2 detected from the membrane. Data show CK2α dose dependent phosphorylation of IKK2 and complete inhibition of this effect by apigenin. B. CK2 Promotes IKK Kinase Activity Catalytically active rCK2α (0.5ng (CK2 0.5ng) or 5ng(CK2 5ng)) and 5ng of rCK2α along with 40μM apigenin (CK2 5ng+Apigenin) respectively were incubated with IKK complex that was immunol precipitated from UM-SCC-6 cell lysate. rCK2α was, then, washed out prior to IKK kinase assay. A fraction of untreated IKK complex was used as negative control (Ctrl). IKK kinase activity was measured with the same substrate as in Fig 4D. Quantified total IKK kinase activity was presented as described in Fig 4D. An 13% and 60% increase of total IKK kinase activities from IKK complex preincubated with 0.5ng and 5ng rCK2α respectively was observed compared to untreated control. 40μM anpigenin completely inhibit the rCK2α induced IKK kinase activity. The detected gel bands and corresponding measurements of the intensities of these bands are shown under the chart and in the table respectively.

UM-SCC-6 Cells were cotransfected with pNF-κB-luccis Luciferase plasmid and one of the pcDNA3-IKK vectors (IKK1–AA=S176A/S180A; IKK1–EE=S176E/S180E; IKK1–KA=K44A, IKK1–WT=wild type; IKK2–AA=S177A/S181A; IKK2–EE=S177E/S181E; IKK2–KA=K44A, IKK2–WT=wild type), as indicated, for 24 hours. pCK is used as a negative control. NF-κB activity is normalized to pCK.. A, Effects of IKK2 on NF-κB reporter activity; B, Effects of IKK1 on NF- κB reporter activity. Both wild type (IKK1–WT, IKK2–WT) and dominant positive (IKK1–EE, IKK2–EE) of IKK1 and IKK2 enhanced the NF-κB reporter activity (IKK1-WT to untreated control p< 0.05, IKK1-EE, IKK2-WT and IKK2-EE to their corresponding untreated control p< 0.001). Kinase dead IKK2–KA inhibited ~65% of the constitutive NF-κB reporter activity (p< 0.001). Dominant negative signal mutant IKK2–AA showed no inhibitory effect on NF-κB activity (p>0.10). Kinase dead IKK1-KA inhibited ~50% of NF-κB activity (p<0.001). Little inhibitory but enhancing effect was observed from kinase dead IKK1–AA (p<0.05).

A. Aberrant NF-κB reporter activity is dependent on heat sensitive components of FBS UM-SCC-6, 9, 11A and 11B cells, as indicated in the figure, were either cultured in MEM with 10% non-heat inactivated FBS (FBS-NHI ■), 10% heat inactivated FBS (FBS-HI ▩), switched to supplemented serum free MEM medium (SFM-new ⬚), or adapted to SFM (SFM- Adp ▥) prior to the transfection for NF-κB reporter activity. The NF-κB reporter activity levels were normalized to the level of their corresponding FBS-NHI. ANOVA analysis and Student-Newman-Keuls analysis comparing each of the treatment groups is shown. A reduction of NF-κB reporter activity was observed from the cells that were cultured under both SFM-new and FBS-HI conditions when compared to that of the cells under FBS-NHI conditions. Adaptation to SFM further decreased NF-κB reporter activity by 95% in UM-SCC-6, 11A and 11B cells and 83% in UM-SCC-9 cells (p<0.01). The level of NF-κB reporter activity from SFM-adp is significantly lower than that of both SFM-new and FBS-HI (p<0.01). No statistically significant difference was detected between SFM-new and FBS-HI. B. Comparison of CK2 kinase activity in cells that were cultured in SFM or FBS. Total cell lysates from UM-SCC-6 cells that were cultured either in SFM (SFM) or FBS (FBS) were used for the CK2 kinase assay. A recombinant active CK2 (Active CK2) was used as positive control. 100 μM Apigenin was applied to separate reactions in parallel to each of the conditions (solid, Apigenin). CK2 activity (open, Ctrl) from the cells that were cultured in SFM was reduced to about 50% of that in FBS (p<0.001). The cell lysates that were treated with apigenin (Apigenin) brought down the corresponding CK2 activity from each of these lysates by ~75% of that FBS and ~50% of that of SFM (p<0.05). C. Comparison of CK2β expression level in cells. UM-SCC-6 cells grown in FBS-NHI or SFM-adp and UM-SCC-9 cells grown in FBS-NHI, as indicated, were compared for their CK2β mRNA levels. CK2β mRNA expression levels were measured as described in Fig 3 F using Quantigene kit method. Data show a statistically significant decrease of CK2β mRNA in both SFM treated UM-SCC-6 cells and UM-SCC-9 cells compared to FBS control UM-SCC-6 cells (p< 0.01 for SFM and p<0.001 for UM-SCC-9 Cells). D. Comparison of IKK kinase activity from the cells that were cultured in SFM or FBS. UM-SCC-6 cells were cultured in SFM or FBS. IKK complexes were IPed by an anti-NEMO antibody from 200 μg total protein of whole cell lysate of each culture. IKK kinase activity was measured with an N-terminal 72 amino acid IκBα-GST fusion protein as substrate and a S32G/S36A mutant of the IκBα-GST fusion protein as negative control. Quantified total IKK kinase activity was presented as a ratio of incorporated ³³-P radio-activity of phosphorylated- IκBα-GST quantified by autoradiography to the quantified total-IκBα-GST protein level on the same gel for total IKK kinase activity, and further normalized to the total protein levels of IKK2} on the same gel. A 60% reduction of the normalized IKK kinase activity from these cells were observed. A recombinant active IKK2 was used as positive control (data not shown). . The detected gel bands and corresponding measurements of the intensities of these bands are shown under the bar graph and in the table respectively.