The Drosophila homolog of human tumor suppressor TSC-22 promotes cellular growth, proliferation, and survival

Xiaodong Wu*, Megumu Yamada-Mabuchi*, Erick J. Morris†, Pradeep Singh Tanwar*, Leonard Dobens*‡, Silvia Gluderer§, Sabina Khan*, Jing Cao*, Hugo Stocker§, Ernst Hafen§, Nick J. Dyson†, and Laurel A. Raftery*¶

*Cutaneous Biology Research Center and †Massachusetts General Hospital Cancer Center, Massachusetts General Hospital/Harvard Medical School, Charlestown, MA 02129; and §Institute of Molecular Systems Biology, Eidgeno¨ssiche Technische Hochschule Zu¨rich, 8093 Zu¨rich, Switzerland

Communicated by Terry L. Orr-Weaver, Massachusetts Institute of Technology, Cambridge, MA, February 1, 2008 (received for review April 17, 2007) TSC22D1, which encodes transforming growth factor ␤-stimulated reverses resistance to genotoxic agents in salivary-gland tumor clone 22 (TSC-22), is thought to be a tumor suppressor because its cell lines (3, 4, 10–13). To date, there is no report of TSC22D1 expression is lost in many glioblastoma, salivary gland, and pros- function in normal epithelia. tate cancers. TSC-22 is the founding member of the TSC-22/DIP/Bun bunched (bun) is the only TDB family in the Drosophila family of leucine zipper transcription factors; its functions have not genome (14). Like other TDB , Bun proteins share been investigated in a multicellular environment. Genetic studies strong sequence similarity with TSC-22 in the DNA-binding and in the model organism Drosophila melanogaster often provide –protein-interaction domains (9, 15–20). Like mamma- fundamental insights into mechanisms disrupted in carcinogenesis, lian TDB , bun encodes large and small isoforms, but little because of the strong evolutionary conservation of molecular is known about the relative functions of the two types of mechanisms between flies and humans. Whereas humans and mice isoforms. have four TSC-22 domain genes with numerous isoforms, Drosoph- bun is required for development, oogenesis and viability (18, ila has only one TSC-22 domain gene, bunched (bun), which 19, 21, 22). We have studied bun function in the follicular encodes both large and small protein isoforms. Surprisingly, Dro- epithelium that overlies each maturing oocyte. Here, bun pre- sophila Bun proteins promote cellular growth and proliferation in vents epithelial cells from being recruited to a migratory fate ovarian follicle cells. Loss of both large isoforms has the strongest (23); the boundary between epithelial and migrating cells is phenotypes, including increased apoptosis. Cultured S2 cells de- regulated through EGF and BMP regulation of bun expression pleted for large Bun isoforms show increased apoptosis and less (24). Expression of vertebrate TSC22D1 is similarly up-regulated frequent cell division, with decreased cell size. Altogether, these by receptor tyrosine kinase signaling and down-regulated by data indicate that Drosophila TSC-22/DIP/Bun proteins are neces- BMP signaling during initiation of feather bud outgrowth in sary for cellular growth, proliferation, and survival both in culture chicken skin (25). Thus, the function of TDB genes as targets of and in an epithelial context. Previous work demonstrated that bun EGF and BMP signaling may be conserved between flies and prevents recruitment of epithelial cells to a migratory fate and, vertebrates. thus, maintains epithelial organization. We speculate that reduced The follicular epithelium is an outstanding model to investi- TSC22D1 expression generally reduces cellular fitness and only gate mechanisms for tissue growth and invasive behavior (2, 26). contributes to carcinogenesis in specific tissue environments. Follicle cells (FCs) proliferate as the epithelium matures during oogenesis stages 1–6 (27). FCs enlarge by endoreplication, a bunched ͉ follicle cells ͉ oogenesis modified cell cycle that replicates DNA without cell division (27, 28) and migrate to new positions before terminal differentiation ancer remains the second leading cause of death in the U.S. (reviewed by refs. 29–31). C(1), despite successful therapeutics against many types of Here, we test for a bun function in proliferation. Surprisingly, tumors. Mechanisms that permit tumors to evade chemotherapy large Bun isoforms promote proliferation, growth, and survival must be identified for further progress. Correlations between of Drosophila cells in epithelia and cultured cells. gene expression and tumor progression suggest candidate tumor suppressors; their mechanisms of action often are tested in Results cultured tumor cells. However, cultured cells lack the complex The hypothesis that human TSC22D1 inhibits cell proliferation biology of tumors in vivo. Recently, genes that promote tumor (4, 10, 13, 32–35) makes the simple prediction that loss of the fly progression and invasion have been identified by using the model homolog would lead to increased cell number. bun expresses six genetic organism Drosophila melanogaster (reviewed in ref. 2). protein isoforms that have the same DNA-binding and leucine The strong conservation of genes and molecular mechanisms zipper domains but can be divided into two size classes [Fig. 1 A between flies and humans makes this an ideal organism to evaluate candidate tumor-suppressor functions in vivo. Human TSC22D1 is a candidate suppressor for several cancers Author contributions: X.W., E.J.M., L.D., N.J.D., and L.A.R. designed research; X.W., M.Y.-M., (3–7). In graded tumors, the lowest levels of TSC22D1 antigen E.J.M., P.S.T., L.D., and J.C. performed research; S.G., H.S., and E.H. contributed new reagents/ analytic tools; X.W., M.Y.-M., E.J.M., L.D., S.K., N.J.D., and L.A.R. analyzed data; and X.W. and is detected in the most aggressive astrocytomas (8). In prostate L.A.R. wrote the paper. biopsies, it is absent from carcinoma cells but still present in The authors declare no conflict of interest. adjacent normal epithelium (5). TSC22D1 encodes two protein ‡Present address: Division of Molecular Biology and Biochemistry, University of Missouri, isoforms that share conserved domains with TSC-22/DIP/Bun Kansas City, MO 64110. (TDB) transcription factors from flies to humans. Functional ¶To whom correspondence should be addressed at: Massachusetts General Hospital-East, tests have focused on overexpression of the smaller isoform, 3rd Floor, Building 149, 13th Street, Charlestown, MA 02129. E-mail: laurel.raftery@ TSC-22 (transforming-growth-factor-␤-stimulated clone-22) (9), cbrc2.mgh.harvard.edu. in cultured cells. The prevailing hypothesis is that loss of This article contains supporting information online at www.pnas.org/cgi/content/full/ TSC22D1 expression allows tumor cells to evade apoptotic or 0800945105/DCSupplemental. antigrowth signals. For example, forced expression of TSC-22 © 2008 by The National Academy of Sciences of the USA

5414–5419 ͉ PNAS ͉ April 8, 2008 ͉ vol. 105 ͉ no. 14 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800945105 Downloaded by guest on September 27, 2021 Fig. 1. bunA is required for normal follicle cell proliferation. (A) Schematic of Bun protein isoforms. Red, shared domain with the DNA binding and leucine zipper domains (white dots). Domains from unique exons are depicted in different colors. BunA and BunF share a large exon that contains two motifs Fig. 2. BunA promotes follicle cell growth. (A and B) A sister pair of (white) found in all large TDB proteins. (B) Wild-type sister clones marked by bun[A-Q578X] mutant clone (GFPϪ) and wild-type clone (2XGFP) at stage 10. absence of GFP (GFPϪ, black region) or intense fluorescence from two copies GFP, green; nuclei, blue. (Scale bar, 100 ␮m.) (A) Merged image. (B) Nuclei of the GFP gene (2XGFP, bright region). (D) Sister clones of a homozygous only. Nuclei in the bunA mutant clone (outlined in white) are more densely bunA (bun[A-Q988X]) mutant clone marked by GFPϪ and a homozygous packed than nuclei in the wild-type sister clone (outlined in yellow). (C) Bar wild-type clone marked by 2XGFP. (Scale bars, 40 ␮m.) (C and E) Cell numbers graphs showing mean sizes of follicle cells mutant for or overexpressing bun. for sister clones, GFPϪ (blue) and 2XGFP (red) sisters are paired in graphs. Cell *, P Ͻ 0.01. Cell sizes are normalized to the respective wild-type (wt) control number is not significantly different for wild-type sisters. (C, P ϭ 0.4). Each for each treatment, which is set to be 1.0. (D and E) Overlay of DNA content bunA mutant clone (blue) has fewer cells than its wild-type sister (red) (E, P ϭ profiles between control wild-type FCs, GFPϩ (blue) and GFPϪ (red) (D)or 0.00007). between bunA mutant (GFPϩ, blue) and wild-type (GFPϪ, red) FCs (E). Wild- type profile includes cells from all oogenesis stages; the 4N, 8N, and 16N populations are from first, second, and third rounds of endoreplication, and supporting information (SI) Fig. S1]. The four small iso- respectively. Most bunA mutant FCs were in the 16N population, indicating that three rounds of endoreplication occurred between clone induction and forms, B–E, range from 189 to 254 aa; each has a unique analysis. N-terminal domain. The two large isoforms, A (1,206 aa) and F (1,097 aa), share most of a large N-terminal domain and share small sequence motifs with mammalian large TDB proteins (36). P ϭ 0.01; data not shown). Thus, bun, particularly the large Bun Previous studies used transposon insertion alleles that disrupt isoforms, promotes FC proliferation. multiple transcripts (e.g. refs. 19 and 23). We obtained lethal Premature exit from mitotic cell cycles can lead to fewer cells. loss-of-function alleles that eliminate the DNA-binding and FCs proliferate until stage 6, when Notch signaling halts mitosis dimerization domains of only the large isoforms (SI Text and ref. and initiates endoreplication cycles (27, 28). Because bun blocks 36). These alleles provided a unique opportunity to investigate Notch signaling during late oogenesis (23), we tested for altered the function of TDB large isoforms in the follicular epithelium, expression of two Notch target genes, cut and emc (38, 39), in where we know the most about bun functions (23, 24). Expres- proliferating bunA mutant FCs (Fig. S2). Expression was unaf- sion of the large A isoform alone rescues null and heteroallelic fected, indicating that large Bun isoforms do not influence cell mutations, suggesting that the small isoforms may be dispensable number through regulation of these Notch targets. (36). Initial characterization of FCs lacking large Bun isoforms Studies of bun function in the adult eye indicate that bun suggested that absence of large isoforms has qualitatively similar, influences photoreceptor size (36), so we examined FC size. Immediately after proliferation, there was no difference in mean but more severe, phenotypes during late oogenesis compared cell size for mutant FCs (n ϭ 16) compared with sister clone with insertional mutations (data not shown). wild-type FCs (data not shown). Because Drosophila cells en- We tested for bun regulation of proliferation using clonal large chiefly by endoreplication (40), we also examined cell size analysis (Fig. 1). Paired sister clones, one composed of homozy- after endoreplication. At stage 10, bunA mutant FCs were gous bun mutant cells and one of wild-type cells, were generated significantly smaller, 200 Ϯ 80 pixels per cell for mutant versus by FLP/FRT-mediated mitotic recombination (37). Control ex- 350 Ϯ 90 for wild type (Fig. 2 A –C and Table S1). We confirmed Ϫ periments generated sister pairs of a wild-type GFP clone and that bunA mutant FCs undergo all three endoreplication cycles ϩ a wild-type GFP clone; each averaged nine cells per clone, a using FACS analysis for DNA content (Fig. 2 D and E). Thus, ratio of 1 (Fig. 1 Band C). If bun were a negative regulator of large Bun isoforms are required for normal growth of FCs. proliferation, then each homozygous bun clone would have more This requirement in FC growth and proliferation was unex- cells than its wild-type sister. For the large isoform-specific allele pected. We speculated that large isoforms might regulate growth bun[A-Q988X], mutant and wild-type sister clones averaged 6 differently from insertional mutations that additionally disrupt and 13 cells per clone, respectively, a ratio of 0.45 (Fig. 1 D and function of small isoforms. To test this, we examined stage-10

E). A lethal transposon insertion with very strong FC pheno- FCs mutant for bun[out14], a semiviable allele that disrupts the BIOLOGY

types, bun[6903] (Fig. S1) (23), gave similar results, means of 11 bunB transcription unit with strong effects on FC morphogenesis DEVELOPMENTAL mutant and 20 wild-type cells per clone, a ratio of 0.55 (n ϭ 7, (Figs. S1 and S3). Both the large BunA isoform and the small

Wu et al. PNAS ͉ April 8, 2008 ͉ vol. 105 ͉ no. 14 ͉ 5415 Downloaded by guest on September 27, 2021 specific to BunA/F. Proliferation rates became markedly differ- ent on day 4, so we assayed for apoptotic cells at this time. Significantly, more TUNEL-positive cells were observed in BunA/F-depleted cultures than in controls (Fig. 3D and Fig. S4), indicating that large Bun isoforms promote cell survival in culture. To determine whether cellular growth was altered by BunA/F depletion, we examined cell size distribution using forward light scatter in flow cytometry. At 12 h, there was no difference in the profile of cell sizes between BunA/F-depleted cultures and controls. A significant shift to smaller sizes for BunA/F-depleted cells was detected at 18 h (Figs. S4C and S5) and became pronounced by 36 h (Fig. 3C), the time at which BunA/F- depletion was detectable by Western blotting (data not shown). More BunA/F-depleted cells were smaller with each additional day, whereas control cells were unchanged. The smallest, apo- ptotic fraction of BunA/F-depleted cells did not increase above that of the control until day 3 (Fig. S4C). Thus, decreased cellular growth preceded the increase in apoptosis. Normal cells balance growth with cell division by coordinating cell cycle progression with the rate of protein synthesis (reviewed in refs. 41 and 42). Reduced cell size could arise from decoupling the cell cycle from cellular growth in either of two ways: an increased rate of cell division without a proportionate increase in growth or a decreased rate of cellular growth without a proportionate decrease in proliferation (43). However, there was Fig. 3. bunA promotes S2 cell growth, division, and survival. Ds RNA- no consistent difference in the proportion of cells in each phase mediated interference was used to deplete BunA and BunF (labeled as BunA) of the cell cycle between BunA/F-depleted cells and controls from S2 cells. (A) Growth curves of BunA/F-depleted cells (blue) and controls (Fig. S4D). If all cell cycle stages are lengthened proportionately, (red). Luc, luciferase dsRNA control. (B) Western blot showing depletion of the cell cycle profile appears normal (e.g., ref. 44). To assess the BunA/F 4 days after dsRNA treatment. (C) Size distribution of BunA/F-depleted overall cell division rate, we labeled cell contents with the cells (blue) and control cells (red) at 12 h, 36 h, and day 5 after addition of fluorophore carboxyfluorescein diacetate succinimidyl ester dsRNA, as determined by forward scatter (FSC). (D) Percentages of TUNEL- (CFSE) (45). CFSE-labeled cytoplasmic contents are divided ϭ positive nuclei between BunA/F-depleted and control cells on day 4, P 0.01. between daughter cells at each division, so that relative rates of (E) Example of carboxyfluorescein diacetate succinimidyl ester (CFSE) fluores- cent intensity in BunA/F-depleted cells (blue) and control cells (red) at day 0 cell division are detected by flow cytometry as decreases in after dye treatment and day 5, when control cells had significantly less dye (P ϭ fluorescence over time (Fig. 3E). BunA/F-depleted and control 0.00006). n ϭ 3 for all experiments. cells had the same fluorescence intensity on day 0, but fluores- cence of control cells was substantially lower than BunA/F- depleted cells on day 5. Thus, BunA/F-depleted cells divide less BunB isoform are expressed in FCs (24). Mutant FCs again were often, indicating that smaller size is not a secondary consequence significantly smaller than sister clone wild-type FCs, 3.6 Ϯ 1.5 of accelerated division. versus 4.7 Ϯ 2.4 arbitrary units (au) respectively (Table S1 and Slow proliferation of BunA/F-depleted S2 cells resulted from Fig. S3A). We next tested the effects of overexpression, using a combination of slower cell division and increased cell death, so clonal expression of Gal4 to generate GFPϩ cells overexpressing we assayed for apoptotic cells in mutant FCs (Fig. 4). With good BunA, BunB, or lacZ (Fig. 2 C and Fig. S3 B–E and Table S1). nutrition, no cell death is observed in epithelial follicle cells after BunB-overexpressing FCs were not significantly different from stage 1 (reviewed in ref. 46), but approximately half the polar nearby wild-type FCs. However, BunA-overexpressing FCs were cells undergo apoptosis during stages 1–4 (47). Consistent with significantly larger than their neighbors, 7.6 Ϯ 2.3 versus 6.8 Ϯ this, wild-type sister clones had TUNELϩ FCs only during stages 2.1 au, respectively. We conclude that large Bun isoforms are 1–5. In contrast, mutant clones had TUNELϩ FCs throughout both necessary and sufficient for normal FC growth. oogenesis (Fig. 4B). Furthermore, 26% of bunA mutant clones We speculated that TDB proteins might function differently in had TUNELϩ FCs (Fig. 4A) compared with only 4% of cultured cells than in intact epithelia. Thus, we tested the wild-type sister clones (data not shown). Control sister clones requirement for large Bun isoforms in a nonadherent Drosophila were indistinguishable (data not shown). Thus, increased apo- cell line, using double-stranded RNA-mediated interference to ptosis likely contributes to reduced proliferation of mutant FCs. deplete BunA and BunF in S2 cells. Double-stranded RNAs As a final test, we overexpressed individual Bun isoforms (dsRNA) were synthesized from two different sequences unique specifically in polar cells. Excess polar cells undergo apoptosis, to the BunA and BunF mRNAs. Control cultures were treated leaving only two in each cluster by stage 5 (47). We overex- with dsRNA synthesized from the firefly luciferase gene, which pressed BunA, BunB, or BunC using upd-Gal4 (48). At stage 5 is not present in the Drosophila genome. A large polypeptide, or later, 13% of polar cell clusters with BunA overexpression had detected with antibody against the shared sequences of Bun at least three cells (Fig. 4D), whereas all control clusters had two isoforms, was significantly reduced after 4 days of treatment with cells. Expression of the small-isoform BunC did not alter polar either BunA/F dsRNA compared with controls (Fig. 3B). cell number, and the small-isoform BunB had a minimal effect Consistent with in vivo results, large isoform-depleted cultures (Fig. 4D). Before stage 5, expression of BunA did not increase proliferated more slowly than control-treated cultures (Fig. 3A). the mean number of cells per cluster, and ectopic mitosis was not Control cell number increased exponentially between days 3 and observed in 69 clusters stained for phosphohistone H3 (data not 5 after dsRNA treatment. In contrast, numbers of BunA/F- shown). Prolonged survival was not due to a change in cell fate, depleted cells increased only slightly during this period. This because only Fas3ϩ cells were counted in this experiment; effect was observed with both dsRNAs, indicating that it is moreover, BunA promotes loss of Fas3 from epithelial FCs

5416 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800945105 Wu et al. Downloaded by guest on September 27, 2021 are also needed. Insertion mutations in the bunB transcription unit, bun[6903] and bun[out14], are associated with reduced cellular proliferation and growth, but do not directly test the requirement for bunB because they may alter splicing of other isoforms. In FCs, overexpression of BunA gave increased FC size, but overexpression of BunB had no significant effect. Notably, BunA overexpression rescues size and viability of putative bun null larvae, suggesting that loss of all small isoforms has no effect on larval cell growth (36). Overexpression of BunA prolonged polar cell survival, whereas the effect of BunB was minimal. In contrast, ectopic BunB, but not BunA, could alter expression of downstream genes during late oogenesis (24). These differences in phenotypes may result from differential expression of different isoforms, or from mechanistic differences in the functions of large and small isoforms. Detailed studies of specific phenotypes are needed to conclusively distinguish the Fig. 4. bunA promotes follicle cell survival. (A and B) Apoptotic cells are functions of large and small isoforms. detected more frequently in bunA loss-of-function clones than in wild-type Mammalian TDB genes, called TSC22D1, TSC22D2, clones. (A) Example of a bun[A-Q988X] mutant clone with TUNELϩ nuclei TSC22D3, and TSC22D4, encode both large and small isoforms, (red). bunA mutant cells lack GFP (green); nuclei are blue. (B) Many more bunA similar to bun (16, 53). All protein isoforms encoded by these mutant clones have TUNELϩ nuclei than do wild-type clones. Bar graphs genes share the same TSC-22 DNA-binding domain and a ϩ indicate the percentage of clones with TUNEL nuclei for bunA clones (blue) leucine zipper motif that permits both homo- and heterodimer- and wild-type sisters (red), by stage of oogenesis; n ϭ 113 for each. (C and D) Overexpression of bunA influences polar cell number. (C) Example of egg ization (16). We expected that bun would have a function similar chamber from a female with upd-Gal4-driven expression of UAS-bunA. Polar to TSC22D1, the most similar mammalian gene by polypeptide cells have high Fas3 levels (green); arrowhead, group of two; arrow, group of sequence and exon/intron structure (14). Despite this similarity, three; nuclei (blue). (D) Effect of expression of bun gene products on polar cell we find that ectopic BunB does not accelerate apoptosis of polar number. Control polar cell clusters all had two cells after stage 4 (n ϭ 64). cells and that cells overexpressing BunB continue to proliferate. Ϸ Survival of excess polar cells after stage 4 was seen in 10% of the clusters This was a surprise, because increased levels of TSC-22 can expressing bunA (n ϭ 31; *, P ϭ 0.01); bunB rarely had excess polar cells after stage 4. (Scale bars, 20 ␮m.) promote cell death in tumor cell lines (10, 32, 33) and promotes cell cycle arrest induced by TGF-␤ or PPAR-␥ (35), the opposite of the proliferation-promoting functions of large bun isoforms in (Fig. S6). Thus, increased BunA levels promote polar cell flies. This apparent paradox may indicate that a different survival, and large Bun isoforms are necessary to promote mammalian TDB protein shares the BunB function, such as Gilz, epithelial FC survival. Tests for genetic interactions with the encoded by TSC22D3 (54). Gilz, promotes lymphocyte survival central regulators of Drosophila apoptosis also indicate that (17) and has functional similarities with BunB (56). endogenous bun antagonizes apoptosis during eye development Studies of TSC22D1 focused on only the small protein isoform, (Fig. S7). TSC-22; the large isoform has not been investigated. In a genetic study of murine TSC22D1, we find similarities to bun growth Discussion phenotypes (M. Guitard, C. E. Dohrmann, T. Soma, L. L. Dobens, These data demonstrate that large Bun isoforms promote cel- J. Brissette, and L.A.R., unpublished work), suggesting that this lular growth in culture and cell autonomously in vivo. This is a gene also shares in vivo functions with bun. Although TSC22D1 is rare phenotype in S2 cells; a dsRNA interference screen found Ϸ suspected to be a tumor-suppressor gene for some tissues (7, 8, 13, that only 4% of Drosophila genes had a cell size, cell cycle, or 32, 55), it is overexpressed in renal cell carcinoma (56). Reduced cell death phenotype (49). Reduced proliferation of large iso- TSC22D1 expression may promote carcinogenesis only when ac- form-depleted S2 cells is a secondary consequence of reduced companied by mutations that suppress apoptosis, such as p53 cellular growth and increased apoptosis. Similarly, FCs lacking mutations, or in tissues where expression of another TDB gene can large Bun isoforms proliferate poorly, in part from increased rescue growth. Alternatively, reduced expression might promote apoptosis. Large isoforms are required for proliferation in the wing primordium and for full size of adult photoreceptors (36). chemotherapeutic resistance by slowing growth and proliferation In adult primordia, slowly growing cells die when they compete (57, 58). with adjacent cells for survival factors (e.g., in ref. 50); it is An emerging concept in tumor biology is that mutations can unknown whether such competition occurs in FCs. both promote carcinogenesis in one context and decrease cel- Large Bun isoforms are important to reach normal adult size lular fitness in others (59). In flies, bun has context-dependent (19), but genetic studies suggest they are not central to the insulin functions that might antagonize tumor progression. In most FCs, receptor or TOR signaling pathways (36). FCs lacking the large bun is dispensable for epithelial morphology; however, bun BunA/F isoforms maintained normal size while proliferating but mutant FCs are sensitized to migration-inducing signals (23) and not during endoreplication. Unlike other genes necessary for display position-dependent loss of monolayer organization (data final FC size (27, 28, 51), BunA/F do not regulate entry into not shown). Perhaps TSC22D1 similarly prevents neoplasia in endocycles via Notch target genes. Importantly, FCs lacking the presence of potentially oncogenic signals. Consistent with BunA/F can complete all three endocycles by stage 10 of this model, overexpression of TSC-22 inhibits anchorage- oogenesis. Oogenesis slows substantially under poor nutrient independent growth in salivary-gland tumor cells (11). The conditions (52), a developmental flexibility that might permit functions of bun in the follicular epithelium provide a framework slowly growing FCs to reach normal size. The growth pathway for evaluating the importance of TSC22D1 in tumor progression.

regulated by bun remains to be identified. BIOLOGY Materials and Methods

This work does not distinguish whether only the large Bun DEVELOPMENTAL isoforms are necessary for FC growth or whether small isoforms Expanded methods are in SI Text.

Wu et al. PNAS ͉ April 8, 2008 ͉ vol. 105 ͉ no. 14 ͉ 5417 Downloaded by guest on September 27, 2021 Fly Culture and Genetics. Flies were cultured on glucose-cornmeal medium, at cells were measured from clones produced for ref. 23. Clones were selected to 25°C unless indicated. Strains and bunA alleles are in SI Text; all others are in avoid regions of curvature or cell migration. Flybase. Ds RNA Interference. DsRNA was made with the dsRNA Synthesis kit (Ambion). UAS–BunC Transgenic Flies. BunC cDNA (clone GH13775; Open Systems) was Genomic DNA from ywflies was prepared with a DNAeasy kit (Qiagen). subcloned into pUAST; transgenic flies made by the Cutaneous Biology Re- Regions from the unique exons for BunA and BunF were amplified by PCR: search Center Transgenic Fly Core. nucleotides 1,000–1,437 and 1,698–2,517 of the bunA cDNA sequence. Prim- ers are in SI Text. Data shown is from dsRNA against nucleotides 1,000–1,437. Clonal Analyses. Loss of function clones induced with FLP/FRT mediated mitotic Ds-RNA treatment of S2 cells followed ref. 62. recombination (37): 2- to 4-day-old females were treated for from 15 min to 1 h at 37°C and, depending on the stage to be analyzed, reared another 2–4 days TUNEL Labeling. S2 cells were plated on Lab-Tek chamber slides (Nunc) for 2 h. before dissection. Clones with multilayered organization were not scored. Sta- S2 cells or ovaries were fixed, permeabilized, blocked with immunostaining ϩ tistical analyses used the paired ttest. GFP mutant clones were made by MARCM buffer containing 3%BSA, and then incubated for 1 hr at 37°C in reaction (60). For overexpression, we used Flip-out Gal4 (24, 61), and statistical analyses solution (In Situ Cell Death Detection kit; Roche). used the unpaired t test. Numerical results are expressed as mean Ϯ SD. FACS Analysis. FACS was performed on a FACSCalibur cytometer (Becton Immunostaining and Cell Size Measurement. Primary antibodies from mouse: Dickinson). For cell cycle analysis we used propidium iodide staining (63). For ␣-GFP (1:200; Invitrogen), anti-Cut (1:20; Developmental Studies Hybridoma CFSE, we used a protocol for T cells (46). FACS of FCs followed refs. 64 and 65. Bank) (DSHB) and anti-Notch (1:30, C17.9C6; DSHB). From rabbit: anti-Emc (1:500; from Y. Jan, University of California, San Francisco), ␣-GFP (1:200; Invitrogen). Anti-Bun#63 is in SI Text. Secondary antibodies were from goat ACKNOWLEDGMENTS. We thank U. Makhija and P. Gomez-del Arco for technical assistance; M. Guitard and K. White for discussions; D. Harrison and labeled with Alexa Fluor 488 or Alexa Fluor 568 (1:200; Invitrogen). DNA (University of Kentucky, Lexington) and Bloomington Stock Center (Bloom- was stained with ToPro3 (1:5,000; Invitrogen). Ovaries were dissociated and ington, IN) for flies; Y. Jan and Developmental Studies Hybridoma Bank (Iowa mounted in Vectashield (Vector Laboratories) for confocal imaging. City, IA) for antibodies. X.W. was supported by a Massachusetts General Stage 6/7: Individual cells were visualized by Alexa Fluor 546-phalloidin Hospital Medical Discovery Fund Fellowship. This work was also supported by (Invitrogen) and the area measured from confocal images by using NIH Image National Institutes of Health Grants R01GM53203 (to N.J.D.) and 2R01- software. Stage 10: the area of each clone was measured in pixels by using GM60501 (to L.A.R.), an American Cancer Society grant (to L.A.R.), and a grant Adobe Photoshop, and divided by the number of cells. Individual bun[out14] from Shiseido Corp., Ltd. of Japan to the Cutaneous Biology Research Center.

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