Ct log DNA ( pmol) P1P2 Supplementary Fig. S1 Standard curve of the PCR amplification efficiency of transcripts from the P1 and P2 promoters PCR amplification efficiency of mRNAs from the P1 and P2 promoters was compared by control reaction using the equal amount of plasmid DNAs encoding each transcript and the primer sets as in Materials and Methods. Standard curve of the amount of DNA (pmol) vs Ct number is shown. ○ ○ ○ ○
Supplementary Fig. S ( h ) ATF3 -tubulin ATF3 SerumMMS (h) GAPDH relative mRNA expression relative protein expression h serum mRNA MMS mRNA serum protein MMS protein Differential response of the P1 and P2 promoters of human ATF3 gene to serum and genotoxic agent MMS HCT116 cells were serum-starved in the presence of 0.5 % serum for 48 h, and then stimulated by 20 % serum, or 100 g / ml MMS. At each time indicated, ATF3 mRNA (□, ■) or protein (○, ●) was measured as in Methods. Data are means of three independent experiments with standard error bars.
hATF3 GGCGGAGGTGGGGTTAGCTTCAGTTGACCAACCATGCCTTGAGGATAA ATTGGATGGGAT mATF3 GGCAGGGGAGGGGTCAGCCTCAGACAACCAATCCTGCCTTGAGGATAA ATTGTGTAGGAC *** * ** ***** *** **** ***** * ****************** * *** hATF3 CAGATGGGAAGATGTGACAAGAAGAGAAATCCTCCTCTATATAGGATGC TCTGCTGTTTC mATF3 CAGACCAGACAAGAGTATG-GAAGAGAGA-- CTCCTCTGAACAGGATCTCCCACAGGGTC **** ** * * **** **** ******* * ***** * * * * hATF3 CTAAGGATTTTCAGCACCTTGCCCCAAAATG mATF3 CCAAGGAGTTCCAGCACTTCATCCCAAAATG * ***** ** *** *** * ********* hATF3 ATTACGTCAGCCTGGGACTGGCAACACGGAGT- AAACGACCGCGCCGCCAGCCTGAGGGC mATF CTGGGATTGGTAACCTGGAGTTAAGCGGGCTCCCTGCCAACGCGAGGG C ****** *** *** ***** ** ** * * * **** * ****** hATF3 TATAAAAGGGGTGATGCAACGCTCTCCAAGCCACAGTCGCACGCAGCC AGGCGCGCACTG mATF3 TTTAAAAGGGGTGATGCAACGCGCTCCCAGCCACAGTCTCACTCAGCG AGACGC-CGC-G * ******************** **** ********** *** **** ** *** * * * hATF3 CACAGCTCTCTTCTCTCGCCGCCGCCCGAGCGCACCCTTCAGCCCGCG CGCCGGCCGTGA mATF3 CACGGTGCTTCCC CAGTGGAGCCAATCGGCTAACCCGCGCTCCGGCA--GA *** * ** * * **** * * * ******* ***** ** hATF3 GTCCTCGGTGCTCGCCCGCCGGCCAGACAAACAGCCCGCCC--- GACCCCGTCCCGACCC mATF3 GTCCTTGGCGCTCGCCCGCCGGCGGGACAGACCACCCGCCTCTGGCC GCTCTCTGGACCC ***** ** ************** **** ** ****** * * * ** ***** hATF3 TGGCCGCCCCGAGCGGAGCCTGGAGCAAAATG mATF3 TGGCCGCCCCGAGCGAAGACTGGAGCAAAATG *************** ** ************* Supplementary Fig. S3 A B Sequence alignment of the 5’ UTR of transcripts from the P1 and P2 promoters of human and mouse Sequence homology of the 5’ UTR of P1 transcripts of human (148 base; GC 47.3 %) and mouse (145 base; 53,1 %) is shown in A, and that of P2 transcripts of human (265 base; GC 69.4 %) and mouse (245 base; GC 66.9%) is shown in B.
% Input P P kb % Input P P kb 0 Pol II LeoI (PAF1) Supplementary Fig. S4 ■ □ L428 ◆ ◇ DAUDI ■ ◆ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■■ □ □□ □ □□□ □ ◆ ◆◆◆◆ ◆ ◆ ◆◇◇ ◇ ◇ ◇ ◇◇ ◆ ◆ ◆ ◆ ◆ ◆ ◆ ◇◇◇ ◇ ◇◇ ◇ ◇ □ □ □ □ □ □ □□ Recruitment of RNA polymerase II onto the ATF3 gene in cancer cells L428 (■, □) or DAUDI ( ◆, ◇ ) cells were assayed for ChIP using anti-RNA polymerase II (upper panel) or anti-Leo1 antibody (lower panel) as in Methods. Immunoprecipitated DNA was measured by quantitative PCR throughout the human ATF3 gene and expressed as per cent of input DNA. Data represent the means of three independent experiments with standard error bars for control IgG (open) and specific antibodies (closed), respectively.
-5 P P kb % Input Panacetyl H3 -5 P P kb % Input H3K4me3 1.0 Supplementary Fig. S5 ■ □ L428 ◆ ◇ DAUDI ■ ■ ■ ■ ■ ■ ■ ■ □ □ □□ □□□□ ■ ■ ■ ■ ■ ■ ■ ■ □ □ □ □ □ □□ □ ◆◆ ◆◆ ◆◆◆◆ ◇ ◇ ◇◇ ◇◇ ◇ ◇ Chromatin modification of the ATF3 gene in Hodgkin RS cells Immunoprecipitated DNA by anti-panacetyl H3 or anti-trimethyl H3K4 antibodies of L428 (■, □) or DAUDI ( ◆, ◇ ) cells was measured by quantitative PCR throughout the human ATF3 gene and expressed as per cent of the input. Data represent the means of three independent experiments with standard error bars for control IgG (open) and specific antibodies (closed), respectively.