Liu, Y et al
Supplementary Information
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Supplemental figures
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Fig. S1 siRNA knockdown of BRMS1 promotes lung cancer cells invasion. H1299 lung cancer cells were transfected with siRNA control or BRMS1. Post-transfection 48 hrs, the invasion capability was quantified as described. Bar graphs show average cell counts. Data are presented as mean ± S.D.
Fig. S2 RelA/p65 mediates TNF-induced BRMS1 methylation. (A) TNF induces BRMS1 methylation. The relative methylation of BRMS1 was analyzed by quantitative MSP in NHBE cells stimulated with TNF at the indicated doses for 4 hrs. (B) H157 V/I cells were treated with or without 5-Aza (5µM) for 4 days. Bisulfite sequencing PCRs were performed in three independent experiments to detect the methylated CG dinucleotides in the BRMS1 promoter. Each CpG dinucleotide bar from the schematic is depicted by a circle, the fill pattern of which indicates the methylated cytosine and the blank indicated the unmethylated cytosine in CpG dinucleotide.
Fig. S3 NF-kB binds to the -kB binding sites on the BRMS1 promoter. (A) EMSAs performed using P33-labeled wild-type or mutant -kB binding sites as probe incubated with NHBE cells nuclear extracts. (B) TNF enhances NF-kB binding to the NF-kB sites I and II. NHBE cells were treated with TNF (40ng/ml) for 15 min. EMSAs performed as described in (B). The relative expression of RelA/p65 in nuclear (NE) and cytoplasmic (CE) extracts was evaluated by Western blot. β-actin and RNA pol II were probed as controls for cytoplasmic and nuclear protein, respectively.
Fig. S4 DNMT-3b participates in RelA/p65 mediated methylation and transcriptional repression of the BRMS1 promoter. (A) RelA/p65 enhances DNMT-1 and -3b mediated BRMS1 transcriptional repression. p65-/- MEFs cells were co-transfected with BRMS1-Luc. reporter and expression vector encoding Flag-tagged RelA/p65, myc-tagged DNMT-1, -3a, -3b or control. Luciferase activity was determined. (B) ChIP analysis was performed at the indicated time points in H157 V/I cells treated with 5-Aza as described above by quantitative PCR specific to the BRMS1 promoter, as well as GAPDH promoter. (C) RelA/p65 induces chromatin-associated DNMT-1 and -3b. 293T cells were treated with TNF (20ng/ml) and harvested at indicated time points. ChIP analysis was performed across the BRMS1 promoter. The cIAP2 and GAPDH promoters were examined as controls.
Fig. S5 The PTD-p65 (38-47) peptide enhances BRMS1 expression (A) Mutation of E39 abolishes The PTD-p65 (38-47) peptide binding to the BRMS1 promoter. In vitro EMSAs were performed using indicated GST-PTD peptides incubated with 32P-labeled -kB binding site II of BRMS1 promoter as the probe. A supershift band was produced by adding antibody against GST. (B) The PTD-p65 (38-47) peptide increases BRMS1 protein expression in NSCLC. BRMS1 protein levels were analyzed by Western blot in H157 cells treated with the indicated PTD peptides (5mg/ml) for 24 hrs. (C) H157 cells were treated with PTD peptides at indicated doses for 24 hrs. The mRNA levels of cIAP2, Bfl1/A1 and IkBa were determined by QRT-PCR. (*) p<0.05 compared to PTD. (D) H157 cells were treated with 5-Aza as described previously. Following 5-Aza removal, cells were treated with indicated peptides (5µg/ml) and TNF (20ng/ml) for additional 24 hrs. ChIP analysis was performed across the BRMS1 and GAPDH promoter.
Fig. S6 Mutating p65 (109-120) peptide failed to block the interaction of RelA/p65 and DNMT-1. (A) HEK 293T cells were treated with PTD alone, PTD-p65 (109-120) wild type peptide or mutant peptide (5µg/ml) for 24 hrs. IPs were performed using anti-DNMT-1 antibody and the presence of RelA/p65 was detected by Western blots. (B) NSCLC cells were treated with the PTD alone, PTD-p65 (109-120) wild type peptide or mutant peptide (5mg/ml) for 24 hrs. The mRNA of BRMS1 was analyzed by quantitative RT-PCR.
Fig. S7 The interaction of RelA/p65 and DNMT-1 is HDAC independent. H157 cells were transfected shRNA HDAC-1, -2, -3 or a scramble shRNA as control. Immunoprecipitations were performed using antibody against DNMT-1 and the presence of RelA/p65 was detected by Western blot.
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