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1 Supporting Information McJunkin et al /pnas SI Text shrn Library Production. puromycin-selectable retrovirus (plm-puro) was cloned by insertion of an MluI site in the multiple cloning site of pmsv, 3 of the EcoRI site; the resulting vector was digested with XhoI and EcoRI, and a SalI- MfeI fragment from psm2 was inserted. Two hundred forty-nine shrn clones in psm2 were obtained from Open iosystems (Huntsville, L). shrns were subcloned into plm-puro by XhoI/MluI digestion. dditionally, five shrns were PR amplified from long oligonucleotides spanning a XhoI/EcoRI shrn fragment containing constant regions as in psm2. These included the control shrns EN and 23. ES ell ulture and Targeting. KH2 ES cells were cultured on irradiated DR4 fibroblast feeder layers on gelatinized plates in M15 media containing LIF. ES cells were targeted by electroporation of RP1 RP2 RP3 Ponceau S shrp shrp3.143 shrp1.253 shen day 5 μg of the targeting construct with 25 μg pggs-flpe by two 4-V/125-μF pulses in a GenePulserII (io-rad, Hercules, ). Protein Sample Preparation. For protein samples from intestinal epithelial cells, sections of ileum were cleaned of connective tissue and cut longitudinally. Tissue was washed in cold PS, then incubated in PS with 2 mm EDT for 3 min at 4. Mixtures were shaken to free clusters of epithelial cells from the submucosa; epithelial cells were spun down and snap frozen in liquid nitrogen. ES, MEF, and intestinal cell pellets were lysed in hot Laemmli buffer. Mouse Rpa3 antibody (M-18, Santa ruz iotechnology) was hybridized overnight at 4 at 1:1 dilution in 5% milk in TS supplemented with.5% Tween-2 and.1% Triton X-1. ntibodies for human RP were RP3: bcam ab58317 and in-house antibodies RP1: p7-9 and RP2: p34-2. dox no dox no shrn day Rpa3 GFP FLP-In vector TG PGK FRT S-p TRE GFP p TG OR unique XhoI/EcoRI restriction sites for shrn subcloning PGK FRT S-p TREtight p Homing cassette in KH2 ES cells ol1 locus NeoR FRT PGK FRT *Hygro R p co-electroporation of FLP-In vector and pggs FLPe Targeted locus TG ol1 locus FRT S-p TRE GFP p PGK FRT *Hygro R p OR TG ol1 locus FRT S-p p PGK FRT *Hygro R p TREtight Fig. S1. TGM-shRpa3-targeted ES cells express GFP and knock down Rpa3 only in the presence of dox. () Immunoblot of RP1, RP2, and RP3 in whole cell extracts from HT116 cells transduced with different hairpins. RP3 knockdown destabilizes all three RP subunits. () Immunoblot of TG-shRpa3-targeted or untargeted Rosa-rtT ES cells. time course over 4 d of dox treatment, and 4 d of subsequent dox withdrawal is shown. () Schematic of FLP-In targeting vectors. Vectors are adapted from [21] to express a fluorescent protein (GFP or ) with an shrn embedded in the context of in the 3 UTR. The cassette features unique cloning sites for rapid shrn subcloning. Upon electroporation into KH2 ES cells, FLPe recombinase catalyzes FRT-site-specific recombination of the targeting vector into the homing cassette downstream of ol1. orrect recombination donates a start codon to the hygromycin-resistance gene. 1of6

2 days on dox DPI nnexinv sh 4.71 day day 6 % nnexinv-positive cells ycycline % nnexinv-dpi-positive cells ycycline sh Fig. S2. Rpa3 knockdown does not cause apoptosis in MEFs. () nnexin V staining on Rosa26-rtT;TtRM transgenic MEFs. () Proportion of nnexin V- positive early apoptotic cells does not increase over a time course of dox treatment in Rosa26-rtT;TtRM transgenic MEFs. () Proportion of nnexin V/DPI double-positive necrotic cells does not increase over a time course of dox treatment in Rosa26-rtT;TRE--shRN transgenic MEFs. 1 Percent survival ll shrpa3;mv-rtt and shrpa3;rosa-rtt MV-rtT;Rosa-rtT;429 (<1 wks,n=1) Rosa-rtT +/+ ;429 (<1 wks,n=7) MV-rtT;Rosa-rtT; or (<1 wks,n=5) Rosa-rtT +/+ ; or (<1wks,n=7) relative weight Littermate controls MV-rtT;Rosa-rtT;429 MV-rtT;Rosa-rtT;429 (survivors) MV-rtT;Rosa-rtT; or MV-rtT;Rosa-rtT; or (survivors) Fig. S3. shrpa3-induced phenotypes vary with respect to transgenic rtt allele(s) and potency of shrns. () Kaplan Meier survival curve of mice following dox treatment at 5 1 wk of age. () Weight change in MV-rtT;Rosa-rtT;shRpa3 mice treated with dox at 5 1 wk of age. ll mice initially lose weight. fter 8 24 d on dox, mice either become moribund, or begin to regain weight and ultimately survive (even with sustained dox treatment). 2of6

3 intestine bone marrow spleen 45.66% 33.47% Rosa-rtT+/+; sh X-mean = 1,69.2 X-mean = 1, % 14.36% Rosa-rtT+/-; sh X-mean = X-mean = bright % 31.75% Rosa-rtT+/-; sh X-mean = X-mean = % 8.42% Rosa-rtT+/-; X-mean = X-mean = bright MV-rtT;Rosa-rtT+/-; sh 31.89% X-mean = % X-mean = MV-rtT;Rosa-rtT+/-; 14.61% X-mean = % X-mean = bright Fig. S4. Rosa-rtT +/+ drives stronger, more penetrant expression than Rosa-rtT +/, and -high cells are depleted in shrpa3 mice after dox treatment relative to shluci controls. right-field and fluorescent images of intestines are shown, as well as histograms of fluorescence in single-cell suspensions from bone marrow and spleen of mice of indicated genotypes. Percentage of cells in -positive gate are noted, as well as the average intensity of fluorescence (x-mean) in gated cells. ll flow cytometry data were processed using FlowJo analysis software (TreeStar). () Mice were treated with dox for 7 d. Flow cytometry was performed on an LSRII cell analyzer (ecton Dickinson). ( and ) Mice were treated with dox for 2 mo. Flow cytometry was performed on a Guava easyyte (Millipore). Most areas of very bright fluorescence in shrpa3 intestine in are connective tissue. 3of6

4 relative liver weight shrn relative spleen weight shrn relative kidney weight shrn relative liver weight relative spleen weight relative kidney weight.1.8. shrn off dox (3 day pulse). shrn off dox (3 day pulse). shrn off dox (3 day pulse) Fig. S5. Total weight loss caused by shrpa3 is accompanied by general weight loss in various organs. ll relative values were calculated as organ weight/body weight before dox treatment. Values shown are mean of measurements from three mice, except off dox and 5-d dox, where n =2. Error bars are SD. () Liver, spleen and kidney weight decrease concomitantly with total body weight in shrpa3 mice. () Mice treated with a 3-d pulse of dox recover normal liver, spleen and kidney weights by 7 d after dox withdrawal. 4of6

5 sh sh cleaved caspase 3 H&E sh Fig. S6. shrpa3 expression reduces rdu incorporation in the intestinal epithelium, but does not induce apoptosis. () rdu staining on intestine sections from Rosa26-rtT +/+ ;TtRM mice treated with dox for 7 d. (rdu antibody U1/75, ccurate hemical). () Mean number of rdu-positive cells in intestine sections from three mice for each shrn. Error bars are SD. () leaved caspase 3-positive cells were negligible in intestine sections from Rosa26-rtT +/+ ;TtRM mice treated with dox for 7 d. Sections from three mice for each of two Rpa3 and one luciferase shrn were examined. Very sparsely labeled cells confirmed effectiveness of staining procedure relative weight MV-rtT;RosartT;429 littermates Fig. S7. TGM-shRpa3 mice regain weight upon dox withdrawal. Relative weight in MV-rtT;Rosa-rtT;TGM-429 mice treated with a 1-d pulse of dox food at 6 wk of age. 5of6

6 Table S1. shrns used in transgenic alleles shgene.start position shrn sequence (XhoI/EcoRI fragment) 429 sh TGGGGTTTTGTGTTGGTGGG GGGTTGGTTTTGTGG GTGTTTGGGGTTTTTTGTTGTGGTT TGGGGTTTTGTGTTGGTGGG GGTTTTTTTTTGTGGG TGTTTGTTTGGGTGTTGTTGTGGTT TGGGGTTTTGTGTTGGTGGG GTGTTTTTTTGTGGG TGTTTTTGGTTTTTTTGTTGTGGTT TGGGGTTTTGTGTTGGTGGG GTGGTTTGTTTGTGG GTGTTTTGGTTGGGGTTGTTGTGGTT Table S2. Transgenic strains used Strain name Genotype Reference TG-429 6;129-ol1a1 tm1(tre-gfp-429)swl This work TG- 6;129-ol1a1 tm1(tre-gfp-)swl This work (sterile founder lines) TG-sh 6;129-ol1a1 tm1(tre-gfp-sh)swl This work, Premsrirut, et al. (1) TtR- 6;129-ol1a1 tm1(tretight-turborfp-)swl This work TtR- 6;129-ol1a1 tm1(tretight-turborfp-)swl This work TtR-sh 6;129-ol1a1 tm1(tretight-turborfp-sh)swl This work Rosa26-rtT-M2 6.g-Gt(ROS)26Sor tm1(rtt*m2)jae Hochedlinger, et al. (2) MV-rtT Tg(rtThMV)4jd Gossen, et al. (3) 1. Premsrirut PK, (211) rapid and scalable system for studying gene function in mice using conditional RN interference. ell 145: Hochedlinger K, Yamada Y, eard, Jaenisch R (25) Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. ell 121: Gossen M, et al. (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268: of6