Table S1. Primer sequences
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1 Table S1. Primer sequences Primers for quantitative PCR Tgf 1 Forward Tgf 1 Reverse Tgf 2Forward Tgf 2Reverse Tgf 3 Forward Tgf 3 Reverse Tgf r1 Forward Tgf r1 Reverse Tgf r2 Forward Tgf r2 Reverse Thbs1 Forward Thbs1 Reverse Acta2 Forward Acta2 Reverse Cnn1 Forward Cnn1 Reverse DH4S114 Forward DH4S114 Reverse Smad6 Forward Smad6 Reverse Ctgf Forward Ctgf Reverse Hmox1 Forward Hmox1 Reverse p21 Forward p21 Reverse Fn1 Forward Fn1 Reverse Ccl2 Forward Ccl2 Reverse Ccl7 Forward Ccl7 Reverse Il6 Forward Il6 Reverse I B Forward I B Reverse RelB Forward RelB Reverse Mt1 Forward Mt1 Reverse Mt2 Forward Mt2 Reverse Sod1 Forward 5'AACCGCACTGTCATTCACC 3' 5'CGCCAA ACTTCTCCA AACC 3' 5'CTCAACACACCAAAGTCCTC 3' 5'ATCAAAACTCCCTCCCTCC 3' 5'ACCAAAGCGACAGACCTCAC 3' 5'TTCCCCTAACCAACCCACAC 3' 5'AACCGCACTGTCATTCACC 3' 5'CGCCAA ACTTCTCCA AACC 3' 5'AAGGAA AAGAAAAGGGCGG 3' 5'GCACACGGTAGCAGTAGA AG 3' 5'TGCCCAAAGGGAGAAAGTCCAGAA 3' 5'CTGAGCAGCACACACAGAAGCATT 3' 5'TTGCTGACAGGATGCAGAAGGAGA 3' 5'TCACAGTTGTGTGCTAGAGGCAGA 3' 5'TTGTGGATGTGACAGCAGCGTTTG 3' 5'TGTATCACATGGCCTGTGTGTGGA 3' 5'AGGCCTTGTTCTGAACTGTTTGCG 3' 5'GCAGCGCACACTGCAATTCTACAT 3' 5'ACCGGGTTACTCCATCAAGGTGTT 3' 5'GCCGCATTGCTATCTGTGGTTGTT 3' 5'GACAGCTTGTGGCAAGTGAA 3' 5'TTCCTCGTGGAAATCTGACC 3' 5'AGGTGATGCTGACAGAGGAACACA 3' 5'ACAGAGAGAAGGCCACATTGGACA 3' 5'TTGTCGCTGTCTTGCACTC3' 5'AGGTTTGGAGACTGGGAGAG3' 5'TTTTGACAACGGGAAGCATTATCAGATAA 3' 5'TGATCAAAACATTTCTCAGCTATTGG 3' 5'TCACCTGCTGCTACTCATTCACCA 3' 5'AAAGGTGCTGAAGACCTTAGGGCA 3' 5'CCCAATGCATCCACATGCTGCTAT 3' 5'TGCTTCTTGGCTCCTAGGTTGGTT 3' 5'ACCACTTCACAAGTCGGAGGCTTA 3' 5'TGGTACTCCAGAAGACCAGAGGAA 3' 5'ATCCTGACCTGGTTTCGCTCTTGT 3' 5'ACACAGTCATCATAGGGCAGCTCA 3' 5'GCGGATTTGCCGAATCAACAAGGA 3' 5'AACACATTGACAGTCACGGGCTCT 3' 5'AGCTTCACCAGATCTCGGAATGGA 3' 5'ACGCTGGGTTGGTCCGATACTATT 3' 5'TGGCCATATCCCTTGAGCCAGAAA 3' 5'TTGTCGGAAGCCTCTTTGCAGATG 3' 5'TGGCGATGAAAGCGGTGTGC 3'
2 Sod1 Reverse Sod2 Forward Sod2 Reverse Gapdh Forward Gapdh Reverse Nox1 Forward Nox1 Reverse Nox2 Forward Nox2 Reverse Nox4 Forward Nox4 Reverse Rac1 Forward Rac1 Reverse Rac2 Forward Rac2 Reverse 5'GCGGCTCCCAGCATTTCCAG 3' 5'GCACATTAACGCGCAGATCA 3' 5'AGCCTCCAGCAACTCTCCTT 3' 5'AACGACCCCTTCATTGACC 3' 5'TGAAGACACCAGTAGACTCC 3' 5'TGGATGGATCTCTCGCTTCT3' 5'TCACCCAAGCTCTCCTCTGT3' 5'CTTTCTCAGGGGTTCCAGTG3' 5'TGCAGTCTATCATCCAAGC3' 5'CCAGAATGAGGATCCCAGAA3' 5'CAGCAGCAGCATGTAGAAGAC3' 5'TATGGGACACAGCTGGACAA3' 5'TTGAGTCCTCGCTGTGTGAC3' 5'CATCAGCTACACCACCAACG3' 5'TCTCGATGGTGTCCTTGTCA3' Primers used in CHIP Tgf 2 promoter Forward Tgf 2 promoter Reverse Tgf 2 promoter Forward Tgf 2 promoter Reverse Tgf 2 promoter Forward Tgf 2 promoter Reverse Mt1 promoter Forward Mt1 promoter Reverse Sod2 promoter Forward Sod2 promoter Reverse Sod2 intron Forward Sod2 intron Reverse 5' CTGCAACAACAGACCTCACTTCCT 3' 5' TCTTTCTTCCCACTGGTCTTTCCC 3' 5' TCGTGGCCACAGAGTCATCACTAA 3' 5' ACACACACACACACACACACACAC 3' 5' ACTGCAAATTCCTCATGCCAGTCG 3' 5' TCTCAGCGCCTTCTCTCTTTCTCT 3' 5' TGGGTTTACGGAGATAGCTGGCTT 3' 5' ACGTTGAAGTCGTGGTGACGCTTA 3' 5' AATTCTGACCAGCAGCAGAGCCTT 3' 5' TGTGCCAAATTGGTAGAGGCCG 3' 5' TGTGGTTGCTGGACCTTTGGAAGA 3' 5' AGGAAATGCTTTCCCAACTGGGCT 3' Primers used for bisulfite sequencing Tgfβ2 BS Forward Tgfβ2 BS Forward 5' AAGAGTTATTTTGGAGTAATTTATTTG 3' 5' ACTACCTACAAAAAAAACCAACCTC 3' Primers used for measurements of mouse telomere Mouse telomere F Mouse telomere R Mouse 36B4 F Mouse 36B4 R 5'CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT3' 5'GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT3' 5'ACTGGTCTAGGACCCGAGAAG3' 5'TCAATGGTGCCTCTGGAGATT3'
3 Figure S1. Upregulation of TGFβtarget genes in the IKKβnull cells. RNA isolated from wild type and Ikkβ / cells were examined by realtime qrtpcr for expression of genes of the TGFβ pathway. Fold change over uninfected cells was calculated based on data from biological triplicates. Results represent the mean values ± SD from at least two independent experiments. **p<.1 and p<.1 was considered significant. Figure S2. Induction of IKKβ loss in vitro. The Ikkβ F/F fibroblasts were infected with AdGFP and AdCre with or without coinfection with AdIKKβ for 48 h. (A) The infected cells were photographed under light and fluorescence microscopy. More than 9% Ikkβ F/F /AdGFP cells were shown to be GFP positive. (B) The Ikkβ F/F, Ikkβ F/F /AdCre (infected for 48 h) and Ikkβ / cells were treated with TNFα for various times; cell lysates were analyzed by Western blotting for IKKβ, IκBα and βactin. The uninfected or infected Ikkβ F/F cells were used for (C) RNA isolation and analyzed by realtime RTPCR for Ikkβ expression, and (D) transfection with κb Luc reporter and βgal expression plasmid. The Luc and βgal activities were examined 24 h after transfection, and the relative Luc activity (versus βgal activity) was calculated. Fold change over uninfected cells was calculated based on data from biological triplicates. (E) After AdCre infection, the Ikkβ F/F /AdCre cells were passage in vitro for various lengths of time and without or with IKKβ reexpression (AdIKKβ infection). RNA was isolated and analyzed by realtime RTPCR for the expression of NFκBtarget genes. Results represent the mean values ± SD from at least two independent experiments. p<.1 was considered significant. Figure S3. Genotyping of Ikkβ F/F /Tgfβr2 F/F /AdGFP and Ikkβ F/F /Tgfβr2 F/F /AdCre fibroblasts. Genomic DNAs were used for PCR genotyping, using primers recognizing the Ikkβ F, Ikkβ Δ, Tgfβr2 F and Tgfβr2 Δ alleles, and primers for Gapdh. The PCR products were examined on agarose gel after electrophoresis.
4 Figure S4. Induction of IKKβ loss leads to progressive ROS elevation. (A) The wild type cells with and without H 2 O 2 (1 um) treatment for 6 h and the Ikkβ(/) cells with or without AdIKKβ infection for 24 h, and (B) the Ikkβ F/F cells with or without AdCre infection, were labeled with CMH 2 DCFDA and analyzed by flow cytometry at 485/53 nm. y axis, cell numbers, x axis, fluorescence intensity. (C) The cells were divided to two groups based on the level of DCF intensity: the DCFhigh (M2) and DCFlow (M1). The percentage of cells in each group was calculated. Results represent the mean values ± SD from at least two independent experiments. *: p<.5 was considered significant. Figure S5. Increased TGFβ target gene expression and migration of the p65null cells. (A) RNA isolated from wild type and p65 / fibroblasts were analyzed by RTPCR for selected genes of the TGFβ pathways. (B) The cells were subjected to in vitro wound healing assays, and healing rate was calculated by comparison of the wound sizes at and 12 h of wounding. Results represent the mean values ± SD from at least two independent experiments. : p <.1 were considered significant. Figure S6. IKKβ loss induces cjun binding and H3K4Me3 modification of the Tgfβ2 promoter (A) Illustration of approximate locations of potential AP1 binding sites in the Tgfβ2 promoter. The Ikkβ F/F and Ikkβ F/F /AdCre cells were subjected to Chromatinimmunoprecipitation (ChIP) assay using antibodies for (B) transcription factors, p65, p5, Nrf2 and cjun, and (C) histone markers, H3K27Me3, H3K9Me2 and H3K4Me3. The precipitates were examined by PCR using primers for various regions of the Tgfβ2 promoter. The data were calculated as percentage of input and represented the mean values ± SD from at least two independent experiments. p <.1 were considered significant.
5 Relative mrna level WT Ikk / ** Chen, et. al. Supplemental Figure 1
6 Relative luc activity Relative mrna Relative Ikk mrna A C D 2 μm AdGFP AdCre AdGFP AdCre AdIKK AdGFP B Ikk F/F B binding siteluc E TNF (h) IKK I B actin control AdCre_3 days AdCre_9 days AdCre_9 days_adikk Ikk F/F /AdCre Ikk / Ikk F/F Ccl2 Ccl7 Il6 I B RelB Chen, et. al. Supplemental Figure 2
7 Ikk F AdGFP Ikk F/F /Tgf r2 F/F AdCre Ikk Tgf r2 F Tgf r2 Gapdh Chen, et. al. Supplemental Figure 3
8 A B Ikk F/F WT WT_H 2 O 2 Ikk (/) Ikk (/)_AdIKK Control AdCre(3 days) AdCre(9 days) C M1 (Low DCFDA) M2 (High DCFDA) WT 73.35% 24.12% WT_H 2 O % 53.32% Ikk (/) 47.5% 5.54% Ikk (/)_AdIKK 87.9% 11.33% Ikk F/F 73.41% 24.2% Ikk F/F _AdCre_3 days 61.14% 36.7% Ikk F/F _AdCre_9 days 47.7% 49.57% Chen, et. al., Supplemental Figure 4
9 Relative mrna Wound closure % A 12 1 WT p65 / B * 4 2 WT p65(/) Chen, et. al., Supplemental Figure 5
10 % of input % of input A B 1 Control AdCre (18 days) Tgf 2 promoter AP1 Tgf 2 promoter AP p65 p5 Nrf2 cjun IgG p65 p5 Nrf2 cjun IgG p65 p5 Nrf2 cjun IgG C 1 control AdCre (18 days) Tgf 2 promoter ** Chen, et. al. Supplemental Figure 6
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Supplemental Table S1. RT-PCR primers used in this study -----------------------------------------------------------------------------------------------------------------------------------------------
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Supplemental Data mrna Protein A kda 75 40 NEO/vector NEO/RNase L RNASE L β-actin RNASE L β-actin B % of control (neo vector), normalized 240 ** 200 160 120 80 40 0 Neo Neo/RNase L RNase L Protein Supplementary
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