Fig. S1. State diagram for contact-inhibition of proliferation Contact-inhibited cells transition to a contact-independent mode of proliferation upon crossing a critical threshold level of growth factor. Insets show representative fluorescence images probed for BrdU incorporation (green) and DAPI (blue) for epithelial clusters in contact-inhibited and contact-independent states of proliferation. Scale bar: 50 m.

Fig. S2. Identical surface density of adhesion-ligand bound to polyacrylamide gels of varying stiffness. (A) Surface density of ColI bound to polyacrylamide gels was probed by immunofluorescence. ColI-coated substrates were sequentially incubated with anti-ColI mouse antibody (Sigma), and 1 m FluoSpheres carboxylate-modified microspheres (Invitrogen) coated with anti-mouse IgG (Sigma). After each incubation step, the substrates were rigorously washed with PBS on the shaker multiple times. Fluorescence images of the microbeads bound to the substrate surface were acquired using the Zeiss Axiovert 200M microscope. Scale bar: 100 m. (B) The relative number of bound antibody-coated microbeads was counted at multiple image fields by using MATLAB. As a negative control, non-ColI-coated polyacrylamide gels were used and only a negligible number of microbeads were detected (data now shown). Error bars, s.d. (n=3−4).

Fig. S3. The effect of substratum compliance on the threshold EGF levels for contact-inhibition of MDCK cells. MDCK cells grown on soft, intermediate, and stiff surfaces were serum starved and stimulated with different doses of EGF. (A) BrdU uptake (green) and DAPI staining (blue) were assessed 16 hours after EGF treatment. The 0.01 and 0.1 ng/ml EGF cases were not conducted for soft gels because spatial patterns in proliferation were evident even on 100 ng/ml EGF. (B) The fraction of interior cells synthesizing DNA is reported relative to the fraction of cells synthesizing DNA in the periphery of clusters. Error bars, s.d. (n=2), *P<0.05. (C) A state diagram of MCF-10A and MDCK cells during matrix stiffening. Substratum stiffening quantitatively shifts the system closer to the contact-independent proliferation in both MCF-10A and MDCK cells. The two non-transformed cell lines exhibit different sensitivity to this perturbation. The shaded area in the graph refers to the range of EGF concentrations explored experimentally. MDCK on soft and stiff surfaces intersect with the transition line at a point outside of the experimentally accessible level of threshold EGF.

Fig. S4. Stiff substrata attenuate the localization of cell-cell adhesion proteins to intercellular contacts. MDCK cells cultured on soft (7 kPa) and stiff (31 kPa) polyacrylamide gels and glass substratum were serum starved and immunostained for ZO-1 (green) and E-cadherin (red). Nuclei were co-stained with Hoechst 33342 (blue). (A) Heat maps show the relative abundance of ZO-1 in the epithelial clusters integrated across the z-stacks. (B) Merged x-y images (top) were generated by averaging the pixel intensity across the z-stacks. White lines in the merged image (pointed by black arrows) indicate the planes for which x-z views (C) were generated. Scale bar: 10 m. The line graphs show the quantification of relative intensity of ZO-1 and E-cadherin staining along the x-axis at a fixed y-position indicated by the white lines in supplementary material Fig. S4B. Arrowheads indicate the location of cell-cell contacts. The nuclear localization of ZO-1 observed on glass substratum closely resembles the ZO-1 pattern observed on stiff polyacrylamide gels (31 kPa). In contrast, on soft gels (7 kPa), growth-arrested cells in the interior of clusters grown exhibit little, if any, nuclear localization of ZO-1. In addition, the fraction of cellular ZO-1 and E-cadherin at cell-cell junctions was significantly diminished on glass substrates, just as on stiff polyacrylamide gels as shown in (C) line graphs. By contrast, on soft gels, these cell adhesion proteins were predominantly localized to cell-cell contacts among the growth-arrested cells in the interior of a cluster. These results demonstrate that although MDCK cells form cell-cell contacts and grow in clusters on all surfaces (soft gels, stiff gels and glass), the localization of ZO-1 and E-cadherin at cell-cell contacts is compromised on stiffer surfaces (both glass and 31 kPa gel) relative to the soft gel (7 kPa).

Fig. S5. Stiff substrata promote a spatially uniform, contact-independent proliferation. (A) BrdU incorporation (green) and DAPI staining (blue) were assessed in serum starved MDCK cells seeded on soft (7 kPa) and stiff (31 kPa) polyacrylamide gels and glass substratum following 16 h of treatment with 100 ng/ml EGF. The graph shows the percentage of peripheral and interior cells undergoing DNA synthesis. Error bars, s.d. (n=2−5). Cells grown on stiff gel and glass substratum exhibited a spatially uniform BrdU incorporation. These results further corroborate the relationship between compromised cell-cell contacts (supplementary material Fig. S4) and spatially uniform, contact-independent proliferation on stiff substrates, regardless of whether the substrate is a gel or glass.

Fig. S6. Substratum stiffness affect cell spreading in the context of multicellular cluster. The mean projected cell area was calculated by dividing the area of a cell cluster by the number of cells in the cluster. (A) The mean projected cell area was quantified for MDCK cells seeded on soft and stiff substrates (n>30 cell clusters). (B) The mean projected cell area was quantified for MDCK cells seeded on soft substratum after treatment with either control or E-cadherin siRNA (n=10 clusters). Error bars indicate s.d.

Fig. S7. Subcellular localization of β-catenin in cell clusters grown on soft and stiff substrates. MDCK cells grown on soft (7 kPa) and stiff (31 kPa) surfaces were serum starved and immunostained for β-catenin (green). Nuclei were co-stained with Hoechst 33342 (blue). (A) Heat maps show the relative abundance of β-catenin in the epithelial clusters integrated across the z-stacks. (B) Merged x-y images (top) were generated by averaging the pixel intensity across the z-stacks. White lines in the merged image (pointed by black arrows) indicate the planes for which x-z views were generated. White arrows indicate cell-cell contacts. Scale bar: 10 m. On the soft substratum, β-catenin was predominantly localized to cell-cell junctions among all cells in the cluster (left column). By contrast, the subcellular localization of ZO-1 exhibits a difference between interior and peripheral cells. ZO-1 was localized to cell-junctions among growth-arrested interior cells and exhibited cytoplasmic and nuclear staining only among peripheral cells in which cell cycle activity was observed (Fig. 3, left column). Thus, unlike β-catenin, the localization of ZO-1 exhibits a difference between interior and peripheral cells, corresponding to the spatial pattern in proliferation on soft substrates. On the stiff substratum where cell cycle activity is uniform through the cluster, a striking nuclear localization of ZO-1 was observed in all cells (Fig. 3, right column). By contrast, only some cells exhibit a shift in β-catenin subcellular localization on stiff substrates, and even in these cells, β-catenin appears to be in a perinuclear region (right column). Thus, although there is some change in the subcellular localization of β-catenin upon matrix stiffening, it is not as strongly correlated to the spatial pattern in proliferation nor is it as profound a change as observed in ZO-1.

Fig. S8. The nuclear localization of ppERK and pAkt is induced by EGF stimulation. The nuclear localization of ppERK and its role in regulating cell cycle activity have been well documented, including its association with the nuclear transcription factors, such as Fos and Elk-1 (Chen et al., 1992). Furthermore, Akt has been shown to translocate to the nucleus upon growth factor stimulation (Meier et al., 1997) and interact with various binding partners, including B23/NPM, which is known to regulate proliferation and cell survival (Lee et al., 2008). Here, we show that EGF stimulation causes nuclear localization of phosphorylated ERK (ppERK) and phosphorylated Akt (pAkt), and that these events are sensitive to the pharmacological inhibitors, PD98059 and LY294002, of the MEK and PI3K pathways, respectively. Serum-starved MCF-10A cells were pretreated for 2 hours with PD98059 (50 M), LY294002 (50 M), or the solvent DMSO and then stimulated with 10 ng/ml EGF or left untreated. (A) Nuclear ppERK and (B) pAkt (green) signals were assessed by immunofluorescence. Nuclei were labeled by DAPI staining. The bar graphs show the relative intensities of ppERK and pAkt per nucleus. Signal intensities are reported relative to the amount of signals in the nucleus of cells treated with 10 ng/ml EGF in the absence of pharmacological inhibitors (i.e. solvent DMSO). Error bars, s.d (n=2). Scale bar, 50 m. Our data show that the extent of both nuclear ppERK and nuclear pAkt substantially increase following 15 minutes of EGF stimulation. Treatment with the pharmacological inhibitors PD98059 and LY294002 ablated nuclear ppERK and nuclear pAkt signals, respectively. This data confirms that the observed nuclear staining is downstream of the classical EGFR−MEK−ERK and EGFR−PI3K−Akt pathways, respectively.