1. Volume 27, Issue 2, Pages 214-227 (July 2007)
IKKα Shields σ, a G2/M Cell Cycle Checkpoint Gene, from Hypermethylation, Preventing Its Silencing 
Feng Zhu, Xiaojun Xia, Bigang Liu, Jianjun Shen, Yuhui Hu, Maria Person, Yinling Hu 
Molecular Cell 
Volume 27, Issue 2, Pages (July 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 IKKα Regulates 14-3-3σ Expression
(A) Levels of σ expression in WT and Ikkα−/− keratinocytes, detected by western blotting with antibodies against σ, p21WAF1, and filaggrin. Adenovirus, introduction of IKKα, IKKβ, and GFP as a control (Cont) to keratinocytes; NS, nonspecific bands.
(B) Levels of IKKα-induced σ and filaggrin in Ikkα−/− keratinocytes, detected by western blotting. β-actin was used as a protein loading control. +IKKα, introduction of IKKα to keratinocytes; 3d (or 5d), cells cultured for 3 days (or 5 days).
(C) Terminal differentiation was not restored by reintroduction of σ in Ikkα−/− keratinocytes, detected by western blotting. NS, nonspecific bands; HA, hemagglutinin A; adenovirus, introduction of σ, GFP (Cont), and IKKα to IKKα keratinocytes.
(D) Induction of p53, p21WAF1, and σ in response to doxorubicin (Dox) treatment, detected by western blotting σ levels (panels 3 and 4) were obtained from the same protein blotting by two exposure times.
(E) Levels of IKKα, σ, p53, and MDM2, detected by western blotting (WB).
(F) Levels of IKKα, σ, and p53 mRNA, detected by northern blotting (NB). GAPDH, mRNA loading control.
(G) Levels of σ mRNA were detected by RT-PCR (RNA decay assay) following treatment with 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) in keratinocytes. +IKKα, introduction of IKKα to Ikkα−/− keratinocytes.
(H) Decay of σ mRNA levels after DRB treatment (see [G]). Ratio between GAPDH and σ mRNA at 0 time point was set as 1 (mean ± SD, n = 3). The error bars indicate SD value. +IKKα, introducing IKKα to Ikkα−/− (−/−) keratinocytes.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2 Ikkα−/− Keratinocytes Have a Defect in the G2/M Phase
(A and B) Cell cycle profiles of WT and Ikkα−/− (−/−) primary cultured keratinocytes analyzed by flow cytometry with MultiCycle software (Phoenix Flow Systems, Inc., San Diego, CA). The G2/M phase was statistically analyzed (t test, mean ± SD, n = 3). The error bars indicate SD value. Control, GFP; IKKα-KA, kinase inactive IKKα (K44A); Dox, doxorubicin; adenovirus, introduction of IKKα, IKKα-KA,14-3-3σ, and GFP to keratinocytes; asterisk, only comparing the G2/M phase between WT and Ikkα−/− keratinocytes.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
14-3-3σ Expression Is Induced by 5-Aza-dC and IKKα in Ikkα−/− Keratinocytes
(A and B) Effects of 5-Aza-dC on σ expression in WT and Ikkα−/− keratinocytes that were treated for 3 days, detected by RT-PCR. GAPDH, loading control.
(C) DNA methylation in the σ gene in Ikkα−/− keratinocytes, detected by methylation-specific PCR. U, unmethylated PCR primers; M, methylated PCR primers. In the bottom panel, the circles represent CG dinucleotides in the σ gene. The cells were infected with adenovirus expressing IKKα for 3 days or treated with 5-Aza-dC for 3 days. Primer, unmethylated and methylated PCR primers.
(D) Expression of σ induced by reintroduction of IKKα in Ikkα−/− (−/−) keratinocytes, detected by RT-PCR. Adenovirus, introduction of IKKα to keratinocytes.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
14-3-3σ CpG Islands Are Hypermethylated in Ikkα−/− Keratinocytes
(A) ClaI digested none of the DNA clones in the pGEM-T vector from WT (W) keratinocytes but digested some of the DNA clones from Ikkα−/− (M) keratinocytes. DNA fragment sizes (bp) are indicated.
(B) DNA sequences of σ from WT and Ikkα−/− (Mut) keratinocytes after DNA was treated with sodium bisulfite. Numbers represent CpG islands. Asterisk, 5-methylcytosines (C).
(C) A summary for σ sequences of 20 clones from WT and 19 clones from Ikkα−/− (Mut) keratinocytes after DNA was treated with sodium bisulfite. Empty circles represent unmethylated CpG islands. Black circles represent methylated CpG islands.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
IKKα Forms a Complex with H3 in the σ Locus in WT Keratinocytes, and σ Is Associated with Trimethyl-H3-K9, Suv39h1, and Dnmt3a in Ikkα−/− Keratinocytes
(A) Distribution of IKKα in cytoplasm and nucleus, detected by immunofluorescent staining. GFP-IKKα was observed on day 3 after adenovirus infection. DAPI, DAPI-stained nuclei; green color, GFP; merge, GFP-IKKα and DAPI.
(B) Association of IKKα with σ, detected by ChIP assay with anti σ and anti β antibodies for immunoprecipatition. IP, immunoprecipitation; M, DNA ladders with DNA sizes denoted on the left side; adenovirus, introducing HA-IKKα and GFP (Cont) to keratinocytes; PCR, σ, PCR primers; PCR, β, PCR primers; −/−, Ikkα−/− keratinocytes; input, PCR quantitative controls.
(C) Levels of phosphorylated H3-Ser10 in the σ locus from WT and Ikkα−/− keratinocytes, analyzed by ChIP assay with anti-H3-Ser10 and anti-H3 antibodies for immunoprecipatition. No antibody, negative control for ChIP assay.
(D) Levels of IKKα and σ in WT and IkkαK44A/K44A primary keratinocytes, detected by western blotting (WB).
(E) Levels of Suv39h1, Dnmt1, Dnmt3a, σ, and GAPDH mRNA in WT and IkkαK44A/K44A primary cultured keratinocytes, detected by RT-PCR.
(F) Levels of Suv39h1, Dnmt1, Dnmt3a, and Dnmt3b, GAPDH mRNA in WT and Ikkα−/− keratinocytes, detected by RT-PCR.
(G and H) Analysis of associated proteins in the σ DNA complex in WT and Ikkα−/− keratinocytes and IKKα-infected Ikkα−/− keratinocytes, detected by ChIP with antibodies against trimethyl-H3-K9, Suv39h1, Dnmt3a, IKKα, H3, and p53 for immunoprecipitation. PCR, σ-1, primers for the σ promoter; PCR, major satellite, positive control for trimethylated H3-K9; NS, nonspecific bands; input, PCR quantitative control; no antibody, negative control for ChIP assay; −/−, Ikkα−/− keratinocytes; adenovirus, introduction of IKKα to keratinocytes.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
WT IKKα Interacts with H3 and Induces σ Expression; IKKα with Point Mutations Does Not Bind to H3 or Induce σ Expression in Keratinocytes
(A) Top panel shows that GST-H3 interacts with HA-IKKα-WT and HA-IKKα-C80, but not with IKKα-LZ− and IKKα-HLH−, detected by GST pull-down assay. Bottom panel shows the expression levels of different forms of HA-IKKα in cells. WB, western blotting.
(B) Interaction of IKKα-WT with H3 and N-terminal H3, detected by GST pull-down assay. Bottom panel shows sizes of H3 and N-terminal H3 proteins. WB, western blotting; aa, amino acid (GST-H3 and GST-N-H3 proteins).
(C) IKKα-WT and IKKα-C80, but not IKKα-LZ−, IKKα-HLH− interacts with H3 in keratinocytes, detected by immunoprecipitation (IP) with an anti-H3 antibody and western blotting with an anti-HA antibody. WB, western blotting; NS, nonspecific bands; adenovirus, introduction of GFP (Cont), IKKα-WT, IKKα-LZ−, IKKα-HLH−, and IKKα-C80 to keratinocytes.
(D) Association of IKKα with H3, H2A, and H4, detected by immunoprecipitation with an anti-HA antibody in keratinocytes overexpressing HA-IKKα. Proteins were stained with SYPRO Ruby protein dye and identified by mass spectrometric analysis.
(E) Association of trimethyl-H3-K9 with the σ DNA in Ikkα−/− keratinocytes overexpressing IKKα-LZ− or IKKα-HLH−, but not in WT keratinocytes, detected by ChIP assay. PCR, σ, and PCR, major satellite, PCR primers; input, PCR quantitative controls; KA/KA, IkkαK44A/K44A keratinocytes; +IKKα, introducing GFP, IKKα-WT, IKKα-LZ−, and IKKα-HLH− to Ikkα−/− keratinocytes; Cont, Ikkα−/− keratinocytes.
(F) Effect of IKKα-WT, IKKα-KA, IKKα-C80, IKKα-LZ−, IKKα-HLH−, and IKKα-KD− on σ expression in keratinocytes, analyzed by western blotting. β-actin, protein loading control; Ikkα−/− and control, keratinocytes with different Ikkα genotypes; +IKKα, introduction of IKKα-WT, IKKα-KA, IKKα-LZ−, IKKα-HLH−, and IKKα-KD− to Ikkα−/− keratinocytes.
(G) Summary of the indicated activities of different forms of IKKα. NS, not shown; ND, not tested. LL/SS, L/R, and F/P, amino acid substitutions; L, leucine; S, serine; R, arginine; F, phenylalanine; P, proline; KD, kinase domain; LZ, leucine zipper motif; HLH, helix-loop-helix domain.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
IKKα Mutants Isolated from SCC Fail to Bind to H3 and Fail to Induce the Expression of σ
(A) The IKKα protein levels in WT skin and SCC, detected by western blotting. WB, western blotting with an anti-IKKα antibody; NS, nonspecific bands.
(B) The mRNA levels of σ, IKKα, and GAPDH in WT skin and SCC, detected by RT-PCR.
(C) Sequence alterations in three isolated IKKα mutants (Mut1, Mut2, and Mut3). aa, amino acid; R/S, T/A, M/V, K/E, S/P, R/K, N/S, Q/K, S/P, Y/C, and E/K, amino acid substitutions; R, arginine; S, serine; T, threonine; A, alanine; M, methionine; V, valine; K, lysine; E, glutamic acid; P, proline; N, asparagine; Q, glutamine; Y, tyrosine; stop, stop codon; bold lowercase, Ikkα mutation.
(D) Interaction between IKKα and H3, detected by using GST pull-down. RT-PCR shows the RNA levels of overexpressed Flag-tagged IKKα including WT, Mut1, Mut2, and Mut3 in cells. Cont, GFP as control; WB, western blotting; actin, loading control for RNA; NS, nonspecific band.
(E) Expression of σ and filaggrin in Ikkα−/− keratinocytes and Ikkα−/− keratinocytes overexpressing WT IKKα and IKKα mutants (Mut1, Mut2, and Mut3) that were tagged with Flag, detected by western blotting with anti σ and filaggrin antibodies. Keratinocytes were infected with adenoviruses expressing different forms of IKKα for 3 days. WT, wild-type keratinocytes; NS, nonspecific bands.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions