Volume 44, Issue 5, Pages 785-796 (December 2011) p50 (NF-κB1) Is an Effector Protein in the Cytotoxic Response to DNA Methylation Damage  Adam M. Schmitt, Clayton D. Crawley, Shijune Kang, David R. Raleigh, Xiaohong Yu, Joshua S. Wahlstrom, David J. Voce, Thomas E. Darga, Ralph R. Weichselbaum, Bakhtiar Yamini  Molecular Cell  Volume 44, Issue 5, Pages 785-796 (December 2011) DOI: 10.1016/j.molcel.2011.09.026 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 44, 785-796DOI: (10.1016/j.molcel.2011.09.026) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 p50 Is Required for Inhibition of NF-κB Activity by SN1-Methylators (A–C) NF-κB (Ig-κB luc)-dependent luciferase assays. Data show mean NF-κB-dependent luciferase, relative to Renilla, normalized to value without TMZ, ±SD of triplicate samples. (A) WT and p65−/− MEFs were pretreated with vehicle or TMZ (16 hr) followed by 10 ng/ml TNFα. ∗p < 0.005. (Inset: immunoblot with anti-p50 or p65). (B) WT and p50−/− MEFs were treated with TMZ or vehicle (16 hr). (Inset: Immunoblot). ∗p < 0.03. (C) p50−/− MEFs cotransfected with pCMV-p50 (p50) or pCMV (vector) and Ig-κB luc were treated with TMZ or vehicle. ∗p < 0.05. (D) WT BclXL or ΔκB BclXL luciferase reporter assays in MEFs treated with 100 μM TMZ or vehicle (mean ± SD of triplicate samples). ∗p < 0.02, relative to untreated. See also Figure S1. Molecular Cell 2011 44, 785-796DOI: (10.1016/j.molcel.2011.09.026) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 SN1-Methylators Block the DNA Binding of p50-Containing NF-κB (A) EMSA with Ig-κB probe. Top panel: MEFs were treated with 100 μM TMZ for the indicated time. Bottom panel: MEFs were treated with TMZ for 16 hr. SC: specific competitor. NS: nonspecific competitor. (B) Top panel: Immunoblot with the indicated antibody. Bottom panel: EMSA of stable transfectants following treatment with 100 μM TMZ or vehicle (16 hr). (C) Nuclear extracts were isolated from cells either unstimulated (lanes 1 and 6) or stimulated with TNFα (30 min). Extracts were treated with 10 μM TMZ for the indicated time (min). Where indicated, 15 ng WT-p50 was given to sh-p50 extracts prior to treatment. Nonspecific (NS) and specific (SC) competitors and p50 supershift (α-p50) were used as shown. The sample was divided following treatment, and EMSA (upper panel) or IB (lower panel) of the indicated lanes was performed. Data demonstrate inhibition of NF-κB DNA binding by TMZ is reconstituted in sh-p50 extract following addition of WT-p50 without loss of p50 protein. (D) qChIP following treatment with vehicle or 100 μM TMZ (16 hr). IP performed with anti-p65 or anti-RNA Pol II and qPCR with BclXL promoter-specific primers. Data represent enrichment of p65 or RNA Pol II, relative to IgG, ±SEM of three independent experiments. ∗p < 0.01, relative to untreated. (E) qPCR of endogenous mRNA in sh-control (c) and sh-p50 (p50) U87 cells treated with 100 μM TMZ or vehicle (16 hr). Data show relative mRNA expression, normalized to untreated, ±SD of triplicate samples from three separate experiments. ∗p < 0.05, relative to untreated. See also Figure S2. Molecular Cell 2011 44, 785-796DOI: (10.1016/j.molcel.2011.09.026) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Loss of p50 Results in Resistance to SN1-Methylator-Induced Apoptosis (A–C) FACS analysis of annexin V binding 72 hr following treatment (mean ± SD of triplicate samples, repeated at least twice with similar results). (A) U87 cells transfected with control, p50- or p65-siRNA were treated with vehicle or 100 μM TMZ. ∗p > 0.3. (B) MEFs were treated with MNU. ∗p < 0.001, relative to vehicle. (C) p50−/− MEFs cotransfected with empty vector (EV) or WT-p50 (p50) and GFP were treated with 100 μM TMZ. Data represent the GFP-positive population. (D and E) Colony-forming assays in WT and p50−/− MEFs (mean ± SD of duplicate samples shown, repeated thrice with similar results). (D) Cells were treated with the indicated concentrations of TMZ. ∗p < 0.03. ∗∗p < 0.005. (E) Cells were pretreated with 20 μM BG (2 hr) and then with TMZ. ∗p < 0.02. (F) Induction of γH2AX foci in sh-c and sh-p50 cells untreated or treated with 100 μM TMZ. Left: representative merged image of γH2AX foci and nuclei. Right: mean γH2AX foci per nucleus, ±SD of ≥200 cells per group. ∗p < 0.0005, relative to untreated. See also Figure S3. Molecular Cell 2011 44, 785-796DOI: (10.1016/j.molcel.2011.09.026) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Inhibition of NF-κB by O6-MeG Is S Phase Dependent (A) Ig-κB luciferase assay in U87 cells serum-starved and treated with vehicle or TMZ (16 hr). (B) Upper panel: FACS analysis of DNA content in U87 cells at the indicated time (hr) following release from double thymidine block. US: unsynchronized cells. Lower panel: diagram of treatment schedule for (C). (C) Ig-κB luciferase assay at indicated time after thymidine release (x axis) following treatment with vehicle (white bars) or 100 μM TMZ (black bars). ∗p < 0.001, relative to vehicle. (D) Ig-κB EMSA in unsynchronized (US) and synchronized cells (0, 2, 4, 6, and 16 hr after release) following treatment with vehicle or 100 μM TMZ. SC: specific competitor, NS: nonspecific competitor. (E) Ig-κB EMSA. 20 nM duplex substrate was administered to nuclear extracts of U87 cells, and EMSA was performed. SC and NS: as above. (F and G) Ig-κB luciferase assay 6 hr after transfection with indicated duplex substrate. (F) U87 cells. ∗p < 0.005, relative to G:C. (G) The indicated cell line was treated with 20 ng duplex. ∗p < 0.05, relative to G:C. (H) Ig-κB luciferase assay in WT and p50−/− MEFs treated with 6-TG (16 hr). ∗p < 0.05, relative to untreated. Luciferase assay data represent mean ± SD of triplicate samples. See also Figure S4. Molecular Cell 2011 44, 785-796DOI: (10.1016/j.molcel.2011.09.026) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Phosphorylation of p50 Mediates Inhibition of NF-κB (A–C) Ig-κB luciferase assays following pretreatment with vehicle or 100 μM TMZ (16 hr) followed by 10 ng/ml TNFα (mean ± SD of triplicate samples). (A) U20S cells stably expressing tet-on WT-ATR and kd-ATR. ∗p < 0.002. (B) ATM+/+ and ATM−/− cells. (C) U87 cells expressing myc-tagged WT or kinase dead Chk1. ∗p < 0.01. (D) CoIP study. U87 cells were treated with vehicle or 100 μM TMZ (16 hr), and nuclear extracts were isolated. Left panel: IP with the indicated antibody and IB with anti-p50 or anti-Chk1. Right panel: immunoblot of input nuclear sample. (E) Chk1 consensus phosphorylation sequence (human p50 S329: underlined). ∗phosphorylated residue. (F) Kinase assay using immunoprecipitated FLAG-tagged WT-Chk1 (WT) or kd-Chk1 (kd) and p50WT (WT) or p50S329A (mt). Autoradiogram (32P) and IB of the same blot shown. (G) Kinase assay using purified recombinant Chk1 and p50. IB was performed with the indicated antibodies, including anti-phospho-S329-p50 (p-p50). Bottom panel: EMSA of the same blot. (H) Kinase assay following incubation with G:C or O6-MeG:T (O6:T) substrate using nuclear extracts from sh-p50 cells supplemented with p50WT or p50S329A (S329A). Chk1 inhibitor, Gö6976: Gö. Autoradiogram (32P) and IB of the same blot shown. (I) In vivo p50 S329 phosphorylation. Left: U87 cells were treated with vehicle or 100 μM TMZ. Right: U87 cells incubated with siRNA (48 hr) were treated with vehicle or 100 μM TMZ (16 hr). Immunoblot was performed with anti-phospho-S329-p50 (p-p50) and the stripped membrane reprobed with anti-p50 and/or anti-Chk1. (J) Ig-κB luciferase assay in p50−/− MEFs cotransfected with Ig-κB luc and either empty vector, WT-p50, or p50S329A. TMZ (0, 25, 100, 250 μM) was given for 16 hr (mean ± SD of triplicate samples shown). ∗p < 0.005, relative to 0 μM TMZ. (K) Ig-κB EMSA of sh-p50 nuclear extracts supplemented with p50WT or p50S329A and treated with duplex substrate as in Figure 5H. SC and NS: specific and nonspecific competitor, respectively. Blot demonstrates that under kinase conditions, O6-MeG:T but not control substrate blocks the DNA binding of p50WT, not p50S329A. (L) Immunoblot (left) and EMSA (right) of p50WT and p50S329D protein. (M) Ig-κB luciferase assay in U87 cells cotransfected with Ig-κB-luc/Renilla and either empty vector (EV), p50WT, p50S329A, or p50S329D (mean ± SD of triplicate samples shown). ∗p < 0.01. See also Figure S5. Molecular Cell 2011 44, 785-796DOI: (10.1016/j.molcel.2011.09.026) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Phosphorylation of p50 Mediates SN1-Methylator-Induced Cytotoxicity (A) qChIP in U87 cells stably expressing GFP-tagged empty vector, p50WT, p50S329A, or p50S329D. Cells were treated with vehicle or 100 μM TMZ for 16 hr, and IP was performed with the indicated antibody. qPCR was performed with BclXL promoter-specific primers. Data represent promoter enrichment of p50 or Histone H1 relative to IgG control ± SEM of three separate experiments. ∗p < 0.05, relative to untreated p50WT sample. Inset: Immunoblot with anti-p50 demonstrates equal expression of GFP-p50 mutants in stable clones. (B) qPCR of endogenous mRNA in WT MEFs and p50−/− stable clones expressing empty vector (EV), p50WT, or p50S329A following treatment with 100 μM TMZ or vehicle (16 hr). Data show mean, normalized to untreated, ± SD of triplicate samples from three experiments. ∗p < 0.05, relative to untreated. (C) Colony-forming assay in WT and p50−/− MEF stable clones treated with TMZ or vehicle. Cells include parental WT and p50−/− MEFs, and p50−/− MEFs expressing empty vector (EV), p50WT, or p50S329A. Data show mean surviving fraction ± SD of duplicate samples, repeated thrice. ∗p < 0.02, relative to similarly treated p50S329A-expressing cells. Lower panel: Immunoblot with anti-p50. Data from stable transfectants are representative of > 1 clone. See also Figure S6. Molecular Cell 2011 44, 785-796DOI: (10.1016/j.molcel.2011.09.026) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 O6-MeG:T Mismatches Sensitize to DNA Damage-Induced Cell Death Colony-formation assays. Cells were untransfected (UT) or transfected with 20 ng duplex substrate (16 hr) and then unirradiated (−IR) or irradiated (+IR). Data for the −IR cells are normalized to −IR UT, and data for +IR are normalized to +IR UT. No change in surviving fraction is caused by 0.5 Gy, and 2 Gy IR causes 25%–30% decrease relative to −IR. Data show mean value ± SD of duplicate samples, repeated at least twice with similar results. (A) U87 cells, treated with 0.5 Gy. ∗p < 0.0005. (B) U87 cells, treated with 2 Gy. ∗p < 0.02. (C) WT and p50−/− MEFs pretreated with 20 μM BG and then treated as in (B). ∗p < 0.002. (D) U87 cells treated with 0.5 Gy IR and either untransfected (UT) or transfected with the indicated duplex substrate. Treatment was administered in the order indicated. ∗p < 0.002, relative to G:C. See also Figure S6. Molecular Cell 2011 44, 785-796DOI: (10.1016/j.molcel.2011.09.026) Copyright © 2011 Elsevier Inc. Terms and Conditions