1 Supplemental Table 1 Sirna and Primer Sequences Used in This Study

Supplemental Table 1 siRNA and primer sequences used in this study. *Silent mutations are indicated by lower case letters. The top 4 are the siRNA sequences. Real time PCR primer sets for POLD4 were MS440 and MS441, and those for POLD1 were MS515 and MS516. MS 525-530 are for detection of CpG islands. The genomic DNA contents were determined by quantitative RT-PCR using primer sets for POLD4 (MS558 and MS559) and POLD1 (MS512-2 and MS513-2). For cloning of the POLD4, and for introducing silent mutations, MS543-2 and MS625, and MS543-2 and MS624 were used, respectively. The 36-mer primer was used for an in vitro DNA replication assay. 1 Supplemental Table 2 Correlation between arm instability and low POLD4 expression Copy number median Copy number median Cytoband Region in low POLD4 in high POLD4 expression group expression group 1p 1-124.2 NO NO 1q 124.2-245.5 NO NO 2p 1-93.4 NO NO 2q 93.4-243 NO NO 3p 1-91.7 NO NO 3q 91.7-199.5 NO NO 4p 1-50.9 NO NO 4q 50.9-191.4 NO NO 5p 1-47.7 amplification NO 5q 47.7-180.6 NO NO 6p 1-60.5 NO NO 6q 60.5-171.0 NO NO 7p 1-58.9 amplification NO 7q 58.9-158.6 NO NO 8p 1-45.2 deletion NO 8q 45.2-146.3 amplification NO 9p 1-50.6 NO NO 9q 50.6-138.4 deletion NO 10p 1-40.3 NO NO 10q 40.3-135.4 NO NO 11p 1-52.9 NO NO 11q 52.9-134.5 NO NO 12p 1-35.4 NO NO 12q 35.4-132.5 NO NO 13q 16.0-114.1 deletion NO 14q 15.6-106.4 amplification NO 15q 17.0-100.3 NO NO 16p 1-38.2 NO NO 16q 38.2-88.8 NO NO 17p 1-22.2 deletion deletion 2 17q 22.2-78.8 NO NO 18p 1-16.1 NO NO 18q 16.1-76.1 NO NO 19p 1-28.5 deletion deletion 19q 28.5-63.8 NO NO 20p 1-27.1 NO NO 20q 27.1-62.4 NO NO 21p 1-12.3 NO NO 21q 12.3-46.9 NO NO 22q 11.8-49.6 NO NO Xp 1-59.4 NO NO Xq 59.4-154.8 NO NO Seventy-four adenocarcinoma cases with gene expression (1) and copy number (2) information were used for the analysis. They were divided into two groups according to POLD4 expression level, with the threshold set at -1 SD of the average (14 and 60 cases). In each case, the median copy numbers of the chromosome arms are compared to the defined threshold (2), and presence or absence of instability is indicated. POLD4-specific amplifications and deletions are highlighted in yellow and light green, respectively. Note that RB1 and TP53 are located in chromosomes 13q and 17p, respectively. To study the correlation between POLD4 and HDAC expression levels, the 149 NSCLC patients (3) were divided into two groups according to POLD4 expression level, with the threshold set at -1 SD of the average. High and low HDAC2 expression levels were judged to determine the average. Fisher’s exact test was used to compare among the groups. 3 Supplemental figure legends Supplemental Figure 1. mRNA expression levels. A. Analysis of microarray data showing mRNA expression levels of the indicated genes. POLA1, catalytic subunit of pol α; ANAPC1, subunit of anaphase promoting complex; ATR, DNA damage checkpoint gene; CDT1 and CDC6, licensing proteins for DNA replication; RAD9A, located 50 kb adjacent to POLD4. B. Analysis of microarray data from GDS1688. Supplemental Figure 2. Hierarchical clustering of 149 NSCLCs, 9 SCLCs, and 5 mixtures of normal lung tissues for the 257 DNA genes involved in DNA metabolism. SCLC and normal lung clusters are indicated. POLD4 is located at the bottom of the list of genes. Supplemental Figure 3. The low level of POLD4 expression found was unlikely due to detection of splicing variants between the SCLC and control samples, as our probe and PCR primers were able to detect the 3’ UTR that was present regardless of the mRNA structure, and the GDS1688 microarray platform included multiple probes for quantification. Shown is an illustration of each detection site on POLD4 splicing variant structures (http://www.h-invitational.jp/). Colors for exons, introns, and UTRs are indicated, with annotation referenced from H-InvDB (JBIRC IDB, Tokyo, Japan). Agilent and Affymetrix, probe sites; Quantitative RT-PCR, PCR target site; WB, epitope site against the monoclonal antibody. Supplemental Figure 4. mRNA levels of POLD4 in a cell line panel. These levels were 4 correlated with the protein expression levels of POLD4 (Fig. 1C in main text). mRNA levels of POLD1 are also shown. Asterisk indicates p <0.05 (t-test). Supplemental Figure 5. A. After treating cells with 0, 1, or 2 μM of 5 aza-dC for 5 days, with the medium change in every 24 hours, total RNA was extracted and quantitative RT-PCR performed. The results were corrected by 18S expression values and are presented as relative to that without drugs, which was set at 1. Bars indicate SD. Asterisks indicate p values (**<0.01, t-test). On the right side, examples of the sequencing patterns in the promoter CpG site are shown. Genomic DNA from ACC-LC-76 was treated with Sodium bisulphate (MethylEasyTM Xceed Rapid DNA Bisulfite Modification Kit, Takara, Japan). The promoter region was amplified by PCR and sequenced using the following sets of primers; MS525 and MS528, MS526 and MS529, MS527 and MS530. An arrowhead indicates the target site. B. POLD4 has been reported to be rapidly degraded by DNA damage (4). Our recent findings of frequent double-stranded DNA breaks (DSB) in SCLC prompted us to measure the amounts POLD4 in the presence of 10 μM of MG132 (left panel), a proteasome inhibitor, as well as the indicated concentrations of caffeine (right panel), an ATM/ATR inhibitor, for 48 hours. Neither of those increased the level of POLD4 in the SCLC cell lines ACC-LC-48 and ACC-LC-172, while MG132 increased that of POLD4 in the NSCLC cell line A549. Supplemental Figure 6. Complementation of POLD4 in SK-LC-6 and ACC-LC-172 cells. A. Two days after retrovirus infection, POLD4 overexpressed cells were sorted by FACS SORP Aria 2 (BD) and used for experiments, with the results 5 shown in Figure 3. B. POLD4 overexpression clones (ACC-LC-172, D4 O/E1 and 2) and their control lines (VC-1, 2, mock) were treated with various concentrations of 4NQO for 48 hours. Live cells were quantitated using MTT assays. Supplemental Figure 7. Effects of POLD4 depletion on cell cycle progression and checkpoint activation. A. Effects of siD4 on DNA contents of randomly cultured cells in the presence or absence of nocodazole (400 ng/ml) for 24 hours. ACC-LC-139 and HCT116 cells were also treated with siRNA and analyzed for DNA contents. B. After treating Calu6 or PC-10 cells with siRNA for 48 hours, mitotic cells were quantified using an Alexa-Fluor 647-conjugated anti-H3-ps28 antibody and propidium iodide. C. After treating Calu6 with siRNA for 48 hours, the cells were analyzed for the expression levels of G1/S checkpoint proteins. Supplemental Figure 8. G1/S checkpoints. A. The knockdown efficiencies of p21 and p27 in Calu6 cells were determined by western blotting analysis. B. After Calu6 cells were treated with the indicated siRNAs for 48 hours, the cell cycle phase was analyzed using 7-amino-actinomycin D (7-AAD, upper panels) or 7-AAD, and an Alexa-Fluor488-conjugated anti-BrdU antibody. C. BrdU incorporation was quantified in each cell cycle phase, as indicated. Interestingly, knockdown of p21 did not abrogate siD4-induced G1 arrest in Calu6 cells. D. Western blotting analysis of p21 was carried out after Calu6 cells were treated with siRNA. The right lanes show Calu6 and A549 cells, which were subjected to 5 Gy of irradiation, then cultured for an additional 6 hours and harvested. In this cell line, the expression level of p21 was intrinsically low and not induced by X-ray irradiation, due to the homozygous deletion of TP53. 6 Therefore, only p27 was employed for G1 arrest. In contrast, p21 may promote the cell cycle in Calu6 cells by enabling assembly of the active kinase complexes of cyclin D with CDK4 and CDK6 (5, 6). E. Western blotting analysis of Calu6 cells to monitor the effect of siDNAPK. F. DNA content was measured after the cells were treated with siDNAPK for 48 hours. Supplemental Figure 9. Total numbers of gaps and breaks. Chromosomal gaps and breaks were quantitated by counting 100 mitotic cells. The total numbers of gaps/breaks were plotted for mock, vector control (VC), and mD4-overexpressed HCT116 cells (pold4). Stable VC and mD4 clones were mixtures of 7 independent clones. Asterisks indicate p values (*<0.05 and **<0.01, Fisher’s exact test). 7 Supplemental references 1. Ding L, Getz G, Wheeler DA, et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 2008; 455: 1069-75. 2. Weir BA, Woo MS, Getz G, et al. Characterizing the cancer genome in lung adenocarcinoma. Nature 2007; 450: 893-8. 3. Takeuchi T, Tomida S, Yatabe Y, et al. Expression profile-defined classification of lung adenocarcinoma shows close relationship with underlying major genetic changes and clinicopathologic behaviors.

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