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Supporting Information Supporting Information Higgins et al. 10.1073/pnas.1210107109 SI Results mL. In vivo analysis was carried out at Southern Research In- At the time of analysis more than 600 ESTs derived from normal stitute using 6-wk-old female NOD/SCID mice from the Na- and diseased tissues and cell lines mapped to the National Center tional Cancer Institute National Laboratory for Cancer for Biotechnology Information unigene cluster Hs. 212395 for Research. Forty mice were assigned randomly to three groups on human Ciz1 (www.ncbi.nlm.nih.gov/sites/entrez?db=unigene). In- day −3. On day −3, 2% sucrose was added to the drinking water dividual ESTs were translated, and the predicted amino acid se- of groups 1 and 2 (15 mice each), and 2 mg/mL doxycycline in quences were compared with full-length Ciz1 with the aim of 2% sucrose was added to the water of group 3 (10 mice). On day 7 verifying the existence of b-variant Ciz1. Ciz1 transcripts that span 0, mice were inoculated s.c. on the right flank with 1.0 × 10 exons 14 and 15 were detected in a total of 39 EST libraries, in- SBC5 ptTS-Neo b-variant shRNA 4 cells (90–95% confluence, cluding 28 that were derived from tumors. Three of five libraries 98.7% viability), in 0.1 mL medium without G418 and hygrom- derived from neuroendocrine lung cancers, 4 of 22 carcinoma li- ycin. On day 21, mice in group 2 were switched to water containing braries, 0 of 3 normal lung libraries, and 1 of 9 libraries derived sucrose and doxycycline. Tumor size was measured twice weekly from other normal tissues or cell lines were represented by b-variant and used to estimate tumor weight. transcripts. Among the lung carcinoma-derived transcripts that span the region, 4 of 10 contained exon 14b, compared with only Imaging. Fluorescence images were collected at 600× magnifi- 1 of 36 noncancer reads. These results show that Ciz1 exon 14 exists cation with a Zeiss Axiovert 200 M microscope fitted with an as two variants (Fig. S1A) and suggest that exon 14b is over- AxioCam camera (Carl Zeiss Vision) and Openlab image ac- represented in lung tumors. Prevalence of b-variant transcripts quisition software (Improvision), using a 63/1.40 oil immersion in lung cancer cells was confirmed using a panel of lung-derived objective and Zeiss filter sets 10, 15, and 2 for Hoescht 33258 cell lines using PCR tools that span the unique exon-junction and (DNA), Alexa Fluor 568, and FITC/GFP, respectively. Constant support selective amplification of transcripts containing exon exposure parameters were used where images give a representa- 14b. Selectivity of junction primer P4 was established using a pair tion of the relative amount of antigen. Images were exported to of sequence-verified Ciz1 clones as pure templates (Fig. S1B). Adobe Photoshop CS2 to allow manipulation of brightness for When applied to cell line-derived cDNAs, this tool generated a reproduction purposes. Identical manipulations were applied so product from neuroendocrine lung cancers but not from a nor- that image sets reflect the relative amount of antigen present. mal fetal lung cell line. In contrast, exon 14a (anchor domain, AD) was detected in all lines, albeit at a lower level in normal cells (Fig. Additional Gene Expression. For cloning, RNA extracted with TRIzol S1C). The majority of the analysis described in this paper exploits (Invitrogen) was reverse transcribed with M-MLV (Promega) and a junction-selective probe (T2, Table S3), rather than a primer set. a 1:1 mix of oligo(dT)/random primers. Ciz1 was amplified using Probe selectivity was established in reactions with two different primers P8/P2, products were subcloned into pGEMTeasy, and primer sets that span exons 14 and 15, using mixtures of Ciz1 DNA from random colonies was digested with EcoR1. Fragment plasmids 20 (exon 14b) and 19 (exon 14a) as template (Fig. S1D), size was visualized by electrophoresis, and insert identity was and was verified in the cellular context by application to a panel of verified by sequencing. Examples of results are shown in Fig. S1F. neuroendocrine lung cancer cell lines. B-variant Ciz1 was elevated For junction-selective PCR, Ciz1 was amplified with junction- dramatically in all small cell lung cancer (SCLC) lines as com- spanning primer P4 coupled with P3. Selectivity was verified pared with three normal lung lines and one neuroendocrine car- using plasmid templates that were isolated from SCLC cell line cinoid (Fig. 3C), and this result was supported by analysis of an SBC2 and cloned in frame into EGFPC3. Sequencing across additional 10 SCLC lines (Fig. S1E). Ciz1 replication domain exon 14 confirmed that clone 20 has exon 14b and that clone 19 (RD) and AD also were elevated in the three tumor lines, but to has exon 14a (Fig. S1B). For quantitative PCR, data were gen- a much lesser extent (Fig. 3C). Similar results also were obtained erated using an ABI 7000, SDS v1.2.3 (Applied Biosystems). usingacloningandsequencing-basedmethodforsixneuroen- Quantitative Western blot analysis was done routinely using docrine lung cancer cell lines, two nonsmall cell lung cancer cell high-throughput precast E-PAGE 8% 48-well gels, Epage sample lines, and three lines derived from normal fetal lung cells (Fig. buffer, and batch-checked, preassembled iBlot dry blotting transfer S1F). In summary, we conclude that exon 14b is overrepresented sandwiches (all Invitrogen). Although only 0.5 μL of plasma is in lung cancer cell lines, that three independent detection meth- required for quantitative detection of b-variant Ciz1, we routinely ods lead to consistent conclusions, and that junction probe T2 is make up 5 μL in a 95-μL loading buffer to minimize possible a selective tool suitable for application to sample sets from pri- inaccuracies in sample loading (10-μL volume). To minimize gel- mary tumors. to-gel variation, complete sample sets are run and transferred on the same day, and all membranes are probed and visualized SI Materials and Methods throughout the process as one, using low exposure of high-per- RNAi. Ten 21-nt siRNAs that span exon14b/exon15 were produced formance chemiluminescence film (GE Healthcare). Residual in vitro (Ambion) or were chemically synthesized (Invitrogen). variation between gels is corrected using a pair of positive and NIH 3T3 cells were transfected with siRNA and/or GFP-hCiz1 negative standards, which are included at both ends of each gel using Mirus 3T3 (Mirus Bio). For inducible delivery of shRNA, to allow assessment of the evenness of protein transfer. Densi- clonal populations of SBC5 cells containing ptTS-Neo and pSIREN- tometry values for variant Ciz1 typically are normalized to en- RetroQ-TetP were generated as described (Clontech). Strains dogenous Ig to control for possible inaccuracies in sample harboring chosen shRNAs were cultured using tetracycline free loading or plasma concentration, as shown in supporting Datasets FBS (Clontech) and were maintained in 100 μg/mL G418, 100 μg/ S1 and S2. This normalization impacts the final outcome, re- mL hygromycin. ShRNAs were induced by doxycycline at 2.5 μg/ ducing the spread of the data and improving ROC AUC values. Higgins et al. www.pnas.org/cgi/content/short/1210107109 1of9 Fig. S1. Validation of b-variant–selective transcript detection tools. (A) Schematic representation of alternative splicing at Ciz1 exon 14 to yield exon 14a or exon 14b. The conditionally excluded region encoding the peptide sequence VEEELCKQ is shown as a red box. Location of primers and probes used in the analysis of Ciz1 expression are shown below, including exon 14b junction-spanning primer P4 and probe T2 (purple). Primer and probe sequences are given in Table S3. (B) Method for analysis of exon 14b expression using exon junction-spanning primer P4 coupled with reverse primer P3. Primer selectivity was verified using plasmid templates. No product is detected when clone 19 is used as template (Lower Left), whereas both plasmids are amplified efficiently by primers P1 and P2 which flank the alternatively spliced region (Lower Right). (C) RT-PCR products generated from cDNA prepared from two neuroendocrine lung cancer cell lines (L95, SBC5) and one normal fetal lung cell line (HFL1), using the b-variant primer set P4/P3 (Top). Total Ciz1 anchor domain (Ciz1 AD) detected with primer set P1/P2 (Middle) and actin detected with P11/P12 (Bottom) are shown also. Products were sequence verified. “No T” is a no-template control. (D) Alternative method for detecting the b-variant transcript using the exon junction-spanning probe T2, illustrated by amplification of mixtures of plasmid clones 19 and 20, at ratios of 100:0, 75:25, 50:50, 25:75, or 0:100. Primers spanning exon 14 (P1/P2, closed symbols, P6/P7, open symbols) that yield a product from both plasmids were coupled with a probe that spans the unique b-variant junction (T2) or which recognizes a region of the AD that is not alternatively spliced (T3 or T4, as indicated). The graph shows that the cycle number required to reach the threshold is constant for nonselective detection tools but is affected bythe composition of the plasmid mixture for the b-variant–selective probe T2. (E) Quantification of b-variant transcript in 10 SCLC cell lines as indicated, detected with primers P1/P2 coupled with probe T2. Data are normalized to actin and calibrated to the result for IMR90 cells, which is given an arbitrary value of one, and are shown with SEM. All SCLC cell lines have elevated b-variant transcript. (F) Example of mobility shift seen in PCR products from clones with exon 14a or 14b.
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