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1 DOI: /ncb2156 Figure S1 Depletion of p114rhogef with different sirnas. Caco-2 (a) and HCE (b) cells were transfected with individual sirnas, pools of the two sirnas or the On Target (OnT) sirna pool. The cells were then fixed and analysed by immunofluorescence with antibodies against the indicated proteins. Note, all p114rhogef specific sirnas induce the same phenotype of warped junctions in Caco-2 cells and disrupted tight junctions in HCE cells. Shown are epifluorescence images. Bars, 10 µm. 1

2 Figure S2 Effect of Rho GEF depletion on junction assembly. (a) Caco-2 cells that had been transfected with sirnas as indicated and plated in low calcium as in figure 2 were incubated in normal medium for 4 hours and then processed for immunofluorescence. Shown are images for the tight junction proteins cingulin and occludin, and the adherens junction proteins α- and β-catenin. Shown are epifluorescence images. (b, c) HCE cells were transfected with sirnas as indicted and, after 3 days, were analysed (b) by immunoblotting for p114rhogef, GEF-H1 and α-tubulin (uncropped images are shown in figure S7) or (c) by immunofluorescence using antibodies against tight junction proteins, ZO-1 and cingulin, and the adherens junction protein β-catenin. Shown are epifluorescence images. (d) Caco-2 cells that had been transfected with sirnas as indicated were fixed and processed for immunofluorescence 24 hours after adding calcium. Shown are epifluorescence images of cells stained for p114rhogef, Myosin IIA and GEF-H1. (e) Depletion of p114rhogef in Caco-2 and MDCK cells. Caco-2 cells were transfected with sirnas and MDCK cells that had been stably transfected with plasmids encoding shrnas were incubated with tetracycline. The cells were incubated for 5 days as in the morphogenesis experiments in figure 3. The cells were then extracted and analysed by immunoblotting. Uncropped images of immunoblots are shown in figure S7. Bars, 10 µm. 2

3 Figure S3 Subcellular localisation of active RhoA. Caco-2 and HCE cells were transfected with the indicated sirnas and, after 2 days, with the RhoA FRET biosensor. RhoA activity was then imaged by gain of CFP fluorescence after acceptor bleaching as in figure 4a. (a) Show are images taken from the apical part of the cells containing the junctional region and from the basal part of the cells. (b, c) The effect of cingulin on junctional RhoA activation was monitored and quantified in the same set of experiments as those shown in figure 4. Show are images of control, p114rhogef and cingulin depleted cells taken from the apical part of the cells that contains the junctional complex (b; see panel a for basal sections). Images were quantified as in figure 4b (c; averages ± 1SD, n: Caco-2, 30 for control 10 for cingulin; HCE, 20 for control and 10 for cingulin). Bars, 10 µm. 3

4 Figure S4 Regulation of junctional myosin activation by p114rhogef. (a) Junction formation by sirna transfected cells was monitored in a Caswitch assay as in figure 2. Shown are epifluorescence images including lateral and basal structures for phosphorylated MLC and f-actin after 1 and 2 hours of junction formation. (b) Effect of p114rhogef depletion on myosin IIA expression and MLC phosphorylation. Caco-2 and HCE cells transfected with control and p114rhogef targeting sirnas were analysed by immunoblotting using antibodies against p114rhogef, myosin IIA, MLC, phosphorylated MLC (ppmlc), and total and phosphorylated myosin light chain phosphatase (MYPT). (c, d) MDCK cells overexpressing active or inactive p114rhogef were cultured as in figure 4e and then lysed and analysed by immunoblotting (c) or processed for immunofluorescence as indicated (d; shown are confocal images from the apical section of the monolayers). (e) Confluent monolayers MDCK cells conditionally expressing p114rhogef-vsv or p114rhogefy260a-vsv were incubated for 16 hours with tetracycline to induce expression prior to analysis by phase contrast microscopy (Bar, 40 µm). Note, the Rock inhibitor Y counteracts the morphological changes induced by p114rhogef overexpression, which also require a functional exchange factor domain. Uncropped images of immunoblots are shown in figure S7. 4

5 Figure S5 The effects on cell shape and junction assembly require an active exchange factor domain. (a) Caco-2 and HCE cells were transfected with cdnas encoding p114rhogef-vsv or p114rhogefy260a-vsv. The cells were then fixed and processed for immunofluorescence. Shown are samples of epifluorescence images that were used for the quantification in figure 4g. (b d) MDCK cells were transiently transfected with sirnas targeting canine p114rhogef. After two days, the cells were transfected with the indicated expression constructs and, after another day in culture, analysed by immunoblotting (b) or immunofluorescence (c, d). Note, only active p114rhogef induces recruitment of junctional markers. Shown are epifluorescence images. Uncropped images of immunoblots are shown in figure S7. Bars, 10 µm. 5

6 Figure S6 Effect of cingulin on junction assembly in HCE and Caco-2 cells. (a, b) HCE cells were transfected with control or individual cingulin sirnas and, after 3 days, were analysed (a) by immunoblotting for cingulin and α-tubulin expression and (c) by immunofluorescence using antibodies against ZO-1 and cingulin. Uncropped images of immunoblots are shown in figure S7 (c) HCE cells were transfected with control, cingulin or p114rhogef sirnas and, after 3 days, were analysed by immunofluorescence using antibodies against GEF- H1. (d) HCE cells were fixed and labelled with antibodies against p114rhogef and cingulin. Shown is a confocal section at the level of tight junctions illustrating co-localisation of the two proteins. (e) Cells were transfected with a cdna encoding myc-tagged cingulin and effects on p114rhogef and myosin IIA distribution were monitored by immunofluorescence. Note, the reduced cytosolic staining of p114rhogef and myosin IIA in cells overexpressing cingulin. (f) Junction formation by sirna transfected Caco-2 cells was monitored in a Ca-switch assay as in figure 2. Shown are stainings for occludin, p114rhogef, myosin IIA and phosphorylated MLC after 1 and 2 hours of junction formation. Panels b, c, e and f show epifluorescence images and panel d confocal sections. Bars, 10 µm. 6

7 Figure S7 Uncropped images of immunoblots. 7