ESM Results:

In vitro assessment of E-cadherin in β-TC6 cell proliferation

In vitro clustering of β-TC6-cells is accompanied by increased E-cadherin levels, reduced cyclin D2 levels, and lower rates of cell proliferation.

To assess the effect of the aggregation of insulin-producing cells on their proliferation, we

generated in vitro islet-like aggregates (pseudo-islets) of SV-40 transformed insulinoma β-TC6

cells. When cultured on gelatine-coated plates, these cells spontaneously reaggregate into 3D

clusters, with a size and shape similar to that of native islets (ESM Fig. 1a). When compared to

monolayer cultures grown in the absence of gelatine coating, β-TC6 pseudo-islets showed

increased levels of E-cadherin protein (ESM Fig. 1b), and less frequent nuclear staining for the

β-catenin protein (ESM Fig. 1c). Quantitative RT-PCR revealed no difference in the expression

of cyclin D1 between monolayers and pseudo-islets (ESM Fig. 1d), but mRNA expression of

cyclin D2 was decreased by more than 60% in the latter preparations (ESM Fig. 1d). This

decrease in cyclin D2 was also reflected at the protein level, as revealed by Western blots by

approximately 3-fold (3.3 ± 1.0 fold, n=2, ESM Fig. 1e). These changes were associated with a

significant decrease in the rate of cell proliferation, as evaluated by EdU incorporation (ESM

Fig. 1f). These data indicate that, in β-TC6 pseudo-islets, the amount of E-cadherin protein

correlates negatively with the levels of cyclin D2 and cell proliferation rates.

In vitro Knock-down of E-cadherin results in increased cyclin D1/D2 levels, which is

accompanied by enhanced cell proliferation.

To assess the direct effects of E-cadherin on D-cyclin levels and cell proliferation, we used an

siRNA that reduced E-cadherin abundance by 90% in monolayers of β-TC6 cells (ESM Fig. 2a).

No change in the transcript abundance of 18 other cadherins was observed (data not shown),

indicating the specificity of the siRNA effect. Three days after exposure to this siRNA, there

were significant increases in mRNA of cyclins D1/D2 (ESM Fig. 2a). A corresponding

approximately 7-fold (6.7 ± 0.3, n=3) increase in cyclin D2 protein was confirmed by Western

blot when the E-cadherin protein was reduced by approximately 80% (-79± 11 %, n=3) (ESM

Fig. 2b). Consistent with this result, the CFSE fluorescence intensity of β-TC6 cells transfected

with the siRNA against E-cadherin was decreased as compared to control (ESM Fig. 2c),

indicating that cell proliferation was 1.5-fold higher in E-cadherin knock-down cells. In contrast,

the siRNA down-regulation of cyclins D1/D2 resulted in a 25% decrease in the proliferation rate

of β-TC6 cells (ESM Fig. 2c). No change in cell proliferation was observed when siRNAs

against E-cadherin plus cyclins D1/D2 were simultaneously transfected into β-TC6 cells (ESM

Fig. 2c). Confirming the CFSE result, cells transfected with siRNA against E-cadherin also

showed a 20% increase in BrDU incorporation (ESM Fig. 2d). These data suggest that the effect

of E-cadherin on cell proliferation was mediated via cyclin D1 and/or D2.

β-catenin regulates the expression of cyclin D1 and c-myc [1], and c-myc regulates the

expression of cyclin D2 [2]. To test the possibility that the reduction in cyclin D levels was due

to reduced binding of β-catenin to the cyclin D1 and c-myc promoters, we performed a

Chromatin Immunoprecipitation (ChIP) assay using an antibody against β-catenin (ESM Fig.

2e). β-TC6 cells stably over-expressing E-cadherin showed decreased levels of β-catenin

binding to the promoter regions of both cyclin D1 (-50%) and c-myc (-40%), as compared to β-

TC6 cells stably transfected with an empty vector (ESM Fig. 2e). These observations indicate

that the changes in beta cell proliferation induced by E-cadherin abundance may be mediated by

alterations in the binding of β-catenin to several target genes.

[1] Li YJ, Wei ZM, Meng YX, Ji XR (2005) Beta-catenin up-regulates the expression of cyclinD1, c-myc and MMP-7 in human pancreatic cancer: relationships with carcinogenesis and metastasis. World J Gastroenterol 11: 2117-2123 [2] Bouchard C, Dittrich O, Kiermaier A, et al. (2001) Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter. Genes Dev 15: 2042-2047