Supporting InformationYang et al. 10.1073/pnas.1420850112SI MethodsDrosophila Stocks and Culture.All crosses were maintained at 25 °C.The UAS/Gal4 system was used for overexpressing genes orknockdown of genes by RNAi constructs. The following strainswere used in this study: (i) ex697 ptc-Gal4 UAS-GFP; (ii) ex697

en-Gal4; (iii) ex697; hh-Gal4 UAS-GFP/TM6B; (iv) nub-Gal4UAS-GFP; (v) en-Gal4; puc-lacZ; (vi) UAS-α-catRNAi [ViennaDrosophila Resource Center (VDRC) no. 107298]; (vii) UAS-α-catRNAi [Transgenic RNAi Project at Harvard Medical School(TRiP) no. 33430]; (viii) UAS-scribRNAi (TRiP no. 29552); (ix)UAS-E-cadRNAi (VDRC no. 27081); (x) UAS-E-cadRNAi (VDRCno. 103962); (xi) UAS-dlgRNAi (VDRC no. 41136); (xii) UAS-lglRNAi (TRiP no. 35773); (xiii) UAS-bskDN; (xiv) ptc-Gal4; (xv)Diap1-GFP 4.3 (gift from J. Jiang, University of Texas South-western, Dallas); (xvi) UAS-exRNAi (VDRC no. 22994); (xvii)UAS-wtsRNAi (VDRC no. 9928); and (xviii) UAS-yki (gift fromD. J. Pan, The Johns Hopkins University, Baltimore).

Immunostaining. Antibody stainings of imaginal discs were per-formed as previously described (1). For Caco-2 cell immunostain-ings, cells were fixed with acetone/methanol (50%/50%) for 10 minor 4% paraformaldehyde for 15 min and then further processedaccording to standard protocols. The following antibodies wereused (source and dilutions in parentheses): mouse anti-βGal(Promega, 1:2,000), rat anti-Drosophila E-Cad [DevelopmentalStudies Hybridoma Bank at University of Iowa (DSHB), 1:30], ratanti-Ci (DHSB, 1:30), rat anti–α-Cat (DSHB, 1:30), mouse anti-Dlg(DSHB, 1:300); guinea pig anti-Dlg (P. J. Bryant, University ofCalifornia, Irvine, CA, 1:1,000), guinea pig anti-Mer (R. Fehon,University of Chicago, Chicago, 1:4,000), mouse anti-Arm (DSHB,1:20), rabbit anti-aPKC (PKCζ, Santa Cruz, 1:500), rabbit anti-cleaved caspase 3 (Cell Signaling, 1:50), rabbit anti-YAP (CellSignaling, 1:25), rabbit anti-YAP (Novus, 1:100), rabbit anti-YAP(Abcam, 1:250), mouse anti-YAP (Abnova, 1:250), mouse anti–E-Cad (BD Bioscience, 1:1,000), mouse anti–E-Cad (Abcam,1:250), sheep anti-hScrib (C-20, Santa Cruz, 1:250), rabbit anti-hScrib (Abcam, 1:250), goat anti-hScrib (Santa Cruz, 1:100),Alexa647-conjugated phalloidin (Invitrogen,1:50), and Alexa568-conjugated phalloidin (Invitrogen,1:50). Images were taken on anOlympus FV1000 confocal microscope.

Mammalian Cell Culture.Caco-2 cells andMDCK cells were culturedin Dulbecco’s Modified Eagle Medium (DMEM, Sigma) con-taining 10% FBS. For calcium depletion experiments, cells weretreated with DMEM containing 10% calcium-depleted FBS.

Generating E-cad, α-cat, and scrib Knockdown Cells. For generatingstable knockdown cells, shRNA containing lentivirus vectors wereacquired from Sigma. Tomake lentivirus, lentiviral constructs andpackaging constructs (ΔVPR and VSV-G) were transfectedinto HEK293T cells together with Fugene (Promega). Lentiviruscontaining supernatants was then collected, filtered, and used toinfect Caco-2 cells. Viral-infected cells were then selected with7 μM puromycin.For siRNA transient knockdown experiments, siRNA stocks

were obtained from Fermentas. We transfected 30 pmol ofsiRNAs into Caco-2 cells together with Lipofectamine RNAi-

MAX (Life Technologies). The transfection media were removedand replenished with DMEM with 10% FBS the next morning.After 24 h, cells were treated with starvation medium (DMEMwithout FBS) for 8 h and subsequently replenished with FBS-containing medium (10% FBS DMEM) for 30 min.For shRNA transfection experiments inMDCK cells, pSUPER

shRNA constructs have been described (2, 3) and were nucleo-fected into MDCK cells. Cells were cultured for 4 d and thenharvested for experiments.shRNA constructs. The following shRNA constructs were used:sh-E-cad, Sigma SHCLNG-NM_004360 CDH1 (E-cad) MIS-SION shRNA; sh-Scrib, Sigma SHCLNG-NM_015356 SCRIBMISSION shRNA; and sh-α-cat, Open biosystem (4).siRNAs (Fermantas).The following siRNAs were used: L-003877–00-0005 ON-TARGETplus CDH1 (E-cad) siRNA, L-010505–00-0005 ON-TARGETplus CTNNA1 (α-Cat) siRNA, and L-010500–00-0020 ON-TARGETplus hSCRIB siRNA.

Quantitative RT-PCR. Total RNA was extracted from Caco-2 cellsusing TRIzol (Invitrogen). After RNA extraction, cDNA wassynthesized using SuperScript First-Strand Synthesis System forRT-PCR (Invitrogen). Primers were used with SYBR SelectMaster Mix (Roche) to perform quantitative RT-PCR reactionsusing a 7900HT real-time PCR system (Applied Biosystems).Primers from the Harvard primer bank (with primer bank ID). The followingprimers were used: hAREG, 22035683c1, (forward primer) GTG-GTGCTGTCGCTCTTGATA, (reverse primer) CCCCAGAAAA-TGGTTCACGCT; CTGF, 98986335c1, (forward primer) CAGC-ATGGACGTTCGTCTG, (reverse primer) AACCACGGTTTG-GTCCTTGG; BIRC3, 342307084c2, (forward primer) TTTCCG-TGGCTCTTATTCAAACT, (reverse primer) GCACAGTGGTA-GGAACTTCTCAT; and GAPDH, 378404907c2, (forward primer)ACAACTTTGGTATCGTGGAAGG, (reverse primer) GCCAT-CACGCCACAGTTTC.Other primers. Other primers used include the following: CTGF,(forward primer) TGGAGATTTTGGGAGTACGG, (reverseprimer) CAGGCTAGAGAAGCAGAGCC; ANKRD1, (forwardprimer) GTGTAGCACCAGATCCATCG, (reverse primer) CG-GTGAGACTGAACCGCTAT; FBN1, (forward primer) ACC-TCGGTGTTGTAAGGTGG, (reverse primer) CCATAAAGG-GCAACCAAGAG; E-cad, (forward primer) GACCGGTGCA-ATCTTCAAA, (reverse primer) TTGACGCCGAGAGCTACAC;α-cat, (forward primer) GAGCTGTCTACGCAAGTCCC, (re-verse primer) TTTCGGAGTACATGGGCAAT; and Scrib, (for-ward primer) AAAGGGGTTGGCAATGACAC, (reverse primer)AGCTGCTCCGCAGTGTG.Primers for MDCK cells. The following primers for MDCK cells wereused: CTGF, (forward primer) CTGAGCATGGAGTGGCAA-GT, (reverse primer) TTTGGCCATTGCTGAAGGCT; AREG,(forward primer) AAGCTGAAGAACGAAAGAAACTTCG,(reverse primer) TGACTCTGATCCGATACCCTGTAA; BIRC3,(forward primer) GCACGAACATCTTTACAAGCAAGA, (re-verse primer) ATGGTGACTGCAGAACTTCCC; and GAPDH,(forward primer) TCATCAACGGGAAGTCCATC, (reverse primer)TTCTCCATGGTGGTGAAGAC.

1. Hamaratoglu F, et al. (2006) The tumour-suppressor genes NF2/Merlin and Expandedact through Hippo signalling to regulate cell proliferation and apoptosis. Nat Cell Biol8(1):27–36.

2. Qin Y, Capaldo C, Gumbiner BM, Macara IG (2005) The mammalian Scribble polarityprotein regulates epithelial cell adhesion and migration through E-cadherin. J Cell Biol171(6):1061–1071.

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3. Capaldo CT, Macara IG (2007) Depletion of E-cadherin disrupts establishment but notmaintenance of cell junctions in Madin-Darby canine kidney epithelial cells. Mol BiolCell 18(1):189–200.

4. Gladden AB, Hebert AM, Schneeberger EE, McClatchey AI (2010) The NF2 tumorsuppressor, Merlin, regulates epidermal development through the establishment ofa junctional polarity complex. Dev Cell 19(5):727–739.

A A’ A’’-Cat

GFP

E-cadGFP E-cad GFP

-Cat GFP

UAS- -catRNAi

dpp-Gal4UAS-GFP

UAS-E-cadRNAi

ptc-Gal4UAS-GFP

TRiP#33430

KK#103962

ptc-Gal4UAS- -catRNAi

wt control

ptc-Gal4UAS- -catRNAi

UAS-E-cadRNAiptc-Gal4

E E’ E’’

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ex-Z GFPGFPex-Z

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ex-Z GFPGFPex-Z

ex-Z

GFP

GFPex-Z

GFPex-Z

Fig. S1. Knockdown of AJs induces Hippo pathway reporter expression. (A and B) Confocal optical cross-sections of third instar wing imaginal discs thatcoexpress UAS-GFP with UAS-α-catRNAi or UAS-E-cadRNAi constructs driven by ptc-Gal4 as indicated. UAS-α-catRNAi and UAS-E-cadRNAi both efficiently knockeddown their targets α-Cat (A′) or E-cad (B′) in the GFP expressing region. (C–F) Full stack confocal images and (G–J) optical cross-sections of wing imaginal discs.(C and G) Expression of UAS-α-catRNAi (TRiP no. 33430) under the control of dpp-Gal4 caused a cell-autonomous decrease and a non–cell-autonomous increaseof ex-lacZ similar to the results from another RNAi transgene (Fig. 1C). (D and H) An alternative RNAi construct for E-cad (KK no. 103962, VDRC) showedinduction of ex-lacZ in a non–cell-autonomous fashion. (E, F, I, and J) Wing imaginal discs that express the Hippo pathway reporter Diap1-GFP and that arestained for Cubitus interruptus (Ci, red) to mark the anterior compartment. E and I show the Diap1-GFP expression pattern in control discs. (F and J) Knockdownof α-cat induced a non–cell-autonomous increase of Diap1-GFP in the posterior compartment.

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UAS-lglRNAi

ptc-Gal4UAS-GFP

UAS-dlgRNAi

ptc-Gal4UAS-GFP

TRiP#35773

TRiP#25780

A A’ A’’Cross-sections

B B’ B’’

C’

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D’’

GFPGFPex-Z

C

D

GFPGFPex-Z

ex-Z

GFP

GFPex-Z

ex-Z

GFP

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ex-Z

ex-Z

Fig. S2. Knockdown of basolateral components induces ex-lacZ expression. Confocal images of third instar wing imaginal discs with genotypes as indicated. (Aand B) Full stack images and (C and D) optical cross-sections. (A and C) Knockdown of lgl induces cell-autonomous expression of ex-lacZ similar to scribknockdown. (B and D) Knockdown of dlg also induces cell-autonomous expression of ex-lacZ.

ptc-Gal4, UAS-GFPUAS- -catRNAi

ptc-Gal4, UAS-GFPUAS- -catRNAi

ArmGFP

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GFP

Arm

GFP

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ptc-Gal4, UAS-GFPUAS- -catRNAi

ptc-Gal4, UAS-GFPUAS-scribRNAi

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MeraPKC

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aPKC

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GFP

Fig. S3. Apical protein localizations are disrupted upon AJ or scrib knockdown. Optical cross-section confocal images of wing imaginal discs that coexpressUAS-GFP with a UAS-α-catRNAi construct (A–C) or a UAS-scribRNAi construct (D) driven by ptc-Gal4. (A) The localization of the AJ component Arm (Drosophilahomolog of β-Cat) is disrupted in α-cat knockdown tissues. (B and C) The apical proteins Crb, aPKC, and Mer are mislocalized in α-cat knockdown cells. (D) Theapical proteins aPKC and Mer are mislocalized in scrib knockdown cells.

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hh-Gal4UAS-GFP

UAS- -catRNAi

UAS-ScribRNAi

hh-Gal4UAS-GFP

hh-Gal4UAS-GFP

UAS- -catRNAi

hh-Gal4UAS-GFP

UAS-bskDN

A A’ A’’

GFPCasp3GFP Casp3

GFPCasp3GFP Casp3

GFPCasp3GFP Casp3

GFPCasp3GFP Casp3

B B’ B’’

C C’ C’’

D D’ D’’

Fig. S4. Induction of apoptosis in α-cat and scrib knockdown tissues. Full stack confocal images of wing discs that coexpress UAS-GFP with different RNAiconstructs as indicated driven by nub-Gal4 in the wing pouch. Discs are stained for cleaved caspase 3 (red in A–D) to mark apoptotic cells. (A) A wild-typecontrol disc showing minimal cleaved caspase 3. (B) An α-cat knockdown disc, where apoptosis was strongly induced and the size of the tissue was reducedcompared with wild type. (C) A scrib knockdown disc is enlarged and has little cleaved caspase 3 staining. (D) Expression of a dominant negative form of theJun kinase bsk (bskDN) in α-cat knockdown cells decreased the amount of cleaved caspase 3 staining and thus blocked cell death and caused an enlargedposterior compartment.

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ptc-Gal4, UAS-GFP ptc-Gal4, UAS-GFPUAS-wtsRNAi UAS- -catRNAi, UAS-wtsRNAi

ptc-Gal4, UAS-GFP ptc-Gal4, UAS-GFPUAS-yki UAS- -catRNAi, UAS-yki

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ex-Z

GFP

-Cat

ex-Z

GFP

-Cat

Fig. S5. Double knockdown of α-cat and wts or overexpression of Yki in α-cat knockdown cells induces cell-autonomous ex-lacZ expression. Optical cross-section confocal images of wing imaginal discs of the indicated genotypes. (A) Knockdown of wts induced cell-autonomous ex-lacZ expression. (B) Simulta-neous knockdown of α-cat andwts induced ex-lacZmainly in a cell-autonomous fashion. (C) Overexpression of Yki strongly induced ex-lacZ. (D) Coexpression ofYki and α-catRNAi induced cell-autonomous ex-lacZ expression.

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A

A’’

A’’’

Dlg

GFP

A’

ptc-Gal4, UAS-GFPUAS- -catRNAi

ex-Z

Fig. S6. Dlg localization is normal in cells adjacent to α-cat knockdown tissue. (A) Optical cross-section confocal images of wing imaginal discs. Yellow arrowsindicate cells next to α-cat knockdown cells. (A′) Normal apical Dlg localization is observed, whereas ex-lacZ expression is significantly increased (A′′).

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controlshRNA

scribshRNA

controlmedium

Calciumdepletedmedium

EcadScrib

DAPI Scrib EcadYAPScribDAPI ScribYAP

EcadScrib

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controlshRNA scrib shRNA

Fig. S7. Knockdown of scrib and calcium depletion activate YAP in Caco-2 cells. (A–D) Cells stained for Scrib (red), E-cad (green), and DAPI (blue). (E–H) Cellsstained for YAP (green), Scrib (red), and DAPI (blue). (A and E) Control cells transfected with a control shRNA. (B and F) Knockdown of scrib did not significantlyaffect E-cad localization, but YAP translocated into the nucleus. (C and G) Cells treated with normal medium. E-Cad and Scrib are localized at the cellmembrane, and YAP is mainly in the cytoplasm. (D and H) Incubating cells in calcium-free medium for 3 h caused internalization of E-cad and Scrib, and YAPwas translocated into the nucleus. (I and J) Quantitative RT-PCR of YAP target genes in Caco-2 cells with (I) scrib shRNA knockdown or (J) cells treated withcalcium-depleted medium, normalized to levels in control cells.

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E-cadshRNA

-catshRNA

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scribshRNA

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ScribYAPDAPI YAP Scrib

ScribYAPDAPI YAP Scrib

ScribYAPDAPI YAP Scrib

I

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nge

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sh-ctrl

sh-scribsh-E-cadsh- -cat

BIRC5

Fig. S8. Knockdown of AJ and basolateral components activates YAP in MDCK cells. (A–D) Cells stained for Scrib (red), E-cad (green), and DAPI (blue). (E–H)Cells stained for YAP (red), Scrib (green), and DAPI (blue). (A and E) shRNA targeting luciferase as a control (ctrl). In control MDCK cells, E-cad and Scrib arelocalized at cell–cell junctions, and YAP is mostly in the cytoplasm. (B and F) Knockdown of α-cat disrupted E-cad and Scrib localization, and YAP translocatedinto the nucleus. (C–G) Similar to α-cat knockdown cells, knockdown of E-cad resulted in Scrib mislocalization and YAP translocation. (D–H) Knockdown of scribdid not affect E-cad localization, but YAP translocated into the nucleus. (I) Quantitative RT-PCR of YAP target genes in MDCK cells with shRNA-mediatedknockdown of control (gray), α-cat (blue), E-cad (green), and scrib (orange), normalized to levels in control cells.

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