SUPPLEMENTAL MATERIAL. Carvajal et al. Supplemental Table 1. List of quantitative PCR primers used for cdna analyses and

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1 UPPLEMEAL MATERIAL Carvajal et al. upplemental Table 1. List of quantitative PCR primers used for cdna analyses and chromatin immunoprecipitation assays. Figure 1. DNA damage-induced transcriptional repression of, CCNB1, CDC2, RRM2, DHFR, and is p5-dependent. (A) Immunoblot analysis of p5 protein levels corresponding to each sample in Figure 1A. (B) Cell cycle profile using flow cytometry of corresponding samples in Figure 1A. (C) Endogenous p5 was downregulated in U2O cells using an sirna approach. Cells were treated with doxorubicin for 24 hr after transfection. amples were harvested 24 hr after treatment with doxorubicin and processed for D-PAGE. Immunoblot analysis shows p5 protein levels before and after DNA damage in either the control or the p5 sirna transfected cells. (B) Quantitative RT-PCR gene expression study of repression targets using samples corresponding to those described in (A). (Error bars =tandard Error of the Mean, D=Days, statistical significance is shown using the student s t-test analysis, p <.1 =, p <.5 =, = not significant.) Figure 2. DNA damage-induced up-regulation of is dose and timedependent. (A) HCT116 cells were treated with increasing amounts of doxorubicin. Cells were harvest at 24 hr post treatment and processed for quantitative RT-PCR analysis using cdna primers specific for, and. (B-C) U2O and MCF7 cells were treated and processed as in (A). (D) U2O cells were treated

2 with.1 µg/ml doxorubicin and harvested at the indicated time points. amples were processed for qrt-pcr expression analysis of,,, DHFR and RRM2. Bar graphs shown are representative experiments. Figure. Ablation of expression followed by DNA damage abrogates p5- dependent transcriptional repression of replication targets, but not mitotic targets. (A) Flow cytometry cell cycle analysis using U2O samples treated as in Figure 5A for the indicated amount of time. (B) Bar graph shows the average of independent experiments as in (A). (C) expression was downregulated in U2O cells using individual sirna oligonucleotides. Control () or (si5,, si7, si8) sirna transfected cells were treated as in Figure 5A. amples were processed for gene expression analysis of, and. (D) expression was targeted as in (C) using a single RNAi oligonucleotide. amples were processed for gene expression analysis of, CDC2, RRM2, DHFR, and E2F8. ( = Nontreated, D= Days, Error bars = tandard Error of the Mean, statistical significance is shown using the student s t-test analysis, p <.1 =, p <.5 =, = not significant). The average of independent experiments is shown. Figure 4.. is not detected at the promoter of the mitotic target gene Purified DNA fragments from the experiment shown in Figure 6D were analyzed for Flag- enrichment at the indicated promoters by quantitative PCR. Figure 5. The cyclin-dependent kinase inhibitor p21 contributes to DNA damageinduced transcriptional repression of p5 targets. Quantitative RT-PCR using mrna extracted from either HCT116 p5+/+, shown in Figure 1A, and p21 -/- cells

3 before and after treatment with the DNA damaging agent doxorubicin. Gene expression analysis of the same genes used in Figure 1A is shown. ( = Non-treated, D= Days, Error bars = tandard Error of the Mean). The average of independent experiments is shown.

4 Carvajal_upplemental Table 1 qrt-pcr Primers Forward Reverse ACCCTGACCTGCTGCTCTT GCCAGGTACTGATGGTCAGTTT E2F6 GGGCCTGCTGCCATCAAAAAT TCATACACTCTCCGCTTTCG GGAAAGGCAACAGCAAACTCT TGGGAGAGCACCAAGAGTAGAAGA E2F8 GCAGCCAATGATACCTCAAAGG ATGAGCACTGCGTGAGAGGGATTA TCCCTGAAAGATCAAGAAGC CCTTGGAAAAATCACCAATC CDK1 AACTAAGAAACCACTTTTCCAT CAATTTTCATCCAAGTTTTGAC CDK2 ATGGAGAACTTCCAAAAGGTGGA CAGGCGGATTTTCTTAAGCG CCNB1 AAGAGCTTTAAACTTTGGTCTGGG CTTTGTAAGTCCTTGATTTACCATG CDC2 CTGGGTTCCTCTGCAGACAT CGAATGTGTCGATCACTGGT DHFR CAGCAGAGAACTCAAGGAAC CCAACTATCCAGACCATGTC RRM2 CAGCAAGCGATGGCATAGT AGCGGGCTTCTGTAATCTGA ACTCTCAGGGTCGAAAACGG CCTCGCGCTTCCAGGACTG TPP5 CTGCCCTGAACAAGATGTTTT CTATCTGAGCAGCGCTCATGG GAPDH CAATGACCCCTTCATTGACC GATCTCGCTCCTGGAAGATG ChIP qpcr Primers AGGAACCGCCGCCGTTGTTCCCGT GCTGCCTGCAAAGTCCCGGCCACT TTCTGGTTTGGCTTCCAGTC GAGGACAGGAAAGCAGATGG (p21) 5' site CTTTCACCATTCCCCTACCC AATAGCCACCAGCCTCTTCT ALB (Albumin) TGGGGTTGACAGAAGAGAAAAGC TACATTGACAAGGTCTTGTGGAG

5 Carvajal_Figure 1 HCT116 A. p5+/+ p5-/- 2D 2D p5 Actin B. HCT116 2D HCT116.7 HCT116.8 HCT116.9 p5+/+ sub 2 4 sub HCT P5-/-.1 sub HCT P5-/ HCT P5-/-.12 p5-/ sub sub 2 4 sub C. U2O sicontrol sirna: sip5 p5 Actin D. normailized to GAPDH U2O RRM sip5 sirna: sicontrol sirna: CDC2 DHFR CCNB1

6 Carvajal_Figure 2 A. HCT116 (p5 +/+) B μg/ml U2O (p5 +/+) μg/ml.15. C. MCF7 (p5 +/+) μg/ml D. Normailized to GAPDH U2O (p5 +/+) Normailized to GAPDH Hrs DHFR RRM Hrs

7 Carvajal_Figure U2O + A. sicontrol U2O.19 sub sub si sub sub 4 sub U2O.2 2 U2O.21 2 U2O.22 D U2O sub U2O % of cells B D ub G2/ M ub G2/ M si sicontrol C. U2O si7 si7 si RRM2.2 DHFR E2F CDC2.4.2 si5 U2O D..2 si5 si si5.2.1 si7 si8

8 Carvajal_Figure 4 H1299 % of Input ALB Transfected: CMV FLAG CMV FLAG IP: anti-flag IP: No Ab

9 Carvajal_Figure 5 normalized to GAPDH HCT D 2D D 2D p21-/1.5 CDK1 CCNB D 2D RRM2.15 2D 2D CDK D 2D D 2D p21-/ 2D 2D

Primers used for PCR of conductin, SGK1 and GAPDH have been described in (Dehner et al,

Primers used for PCR of conductin, SGK1 and GAPDH have been described in (Dehner et al, Supplementary METHODS Flow Cytometry (FACS) For FACS analysis, trypsinized cells were fixed in ethanol, rehydrated in PBS and treated with 40μg/ml propidium iodide and 10μ/ml RNase for 30 min at room temperature.