1. Volume 54, Issue 6, Pages 987-998 (June 2014)
E-Cadherin Couples Death Receptors to the Cytoskeleton to Regulate Apoptosis 
Min Lu, Scot Marsters, Xiaofen Ye, Elizabeth Luis, Lino Gonzalez, Avi Ashkenazi 
Molecular Cell 
Volume 54, Issue 6, Pages (June 2014)
DOI: /j.molcel
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2014 54, 987-998DOI: (10.1016/j.molcel.2014.04.029)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 EMT Attenuates Apoptotic Signaling via DR4 and DR5
(A) NCI-H358 cells were treated with TGF-β (2 ng/ml) for 2 weeks. Parental (P) and mesenchymal (M) cells were fixed, stained with E-cadherin antibody (green), vimentin antibody (red), and DAPI, and analyzed by confocal microscopy. Scale bar, 20 μm.
(B) NCI-H358 cells were stimulated with Apo2L/TRAIL for the indicated time, lysed, and analyzed by IB. cC8, cleaved caspase-8; cC3, cleaved caspase-3.
(C) NCI-H358 cells were stimulated with Apo2L/TRAIL for 2 hr and assayed for caspase-8 and caspase-3 or -7 activity. Values are means ± SD of triplicates.
(D) NCI-H358 cells were stimulated with Apo2L/TRAIL for 8 hr. Cells were harvested, stained for DNA, and analyzed by fluorescence-activated cell sorting (FACS) for sub-G1 DNA content. Values (means ± SD of duplicates) are subtracted from those without Apo2L/TRAIL stimulation.
(E) HCC827 and HCC4006 cells were treated with increasing concentrations of erlotinib (Tarceva; 0.01–4 μM) to generate a resistant (TR) population. Parental (P) and TR cells were lysed and analyzed by IB.
(F) HCC827 cells were stimulated with Apo2L/TRAIL for 72 hr and assayed for cell viability. Values are means ± SD of triplicates.
(G) HCC4006 cells were treated and assayed as in (F). See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Loss of E-Cadherin Function Decreases Apoptotic Signaling via DR4 and DR5
(A and B) We transfected C170 (A) and PATU8902 (B) cells with control siRNA or siRNAs against E-cadherin for 48 hr, followed by Apo2L/TRAIL stimulation for 48 hr and analysis of cell death. Values are means ± SD of triplicates.
(C and D) We transfected C170 (C) and PATU8902 (D) cells with control siRNA or siRNAs against E-cadherin for 48 hr, followed by Apo2L/TRAIL (30 ng/ml for C170 and 100 ng/ml for PATU8902) stimulation for the indicated times and analysis of cell death. Values are means ± SD of triplicates.
(E) C170 cells were transfected with control siRNA (C) or siRNAs against E-cadherin (E) for 48 hr. Cells were stimulated with Apo2L/TRAIL for the indicated time, lysed, and analyzed by IB.
(F) C170 cells were transfected with control siRNA or siRNAs against E-cadherin for 48 hr. Cells were stimulated with Apo2L/TRAIL for 2 hr and assayed for caspase-8 and caspase-3 or -7 activity. Values are means ± SD of triplicates.
(G) C170 cells were treated with E-cadherin-blocking antibody (10 μg/ml) or isotype-matched control antibody for 6 hr. Cells were fixed, stained with E-cadherin antibody (red), β-catenin antibody (green), and DAPI, and analyzed by confocal microscopy. Scale bar, 20 μm.
(H and I) C170 cells were treated as in (G), followed by Apo2L/TRAIL stimulation. Cells were assayed for caspase-8 and caspase-3 or -7 activity at 2 hr (H) and for cell death at 24 hr (I). Values are means ± SD of triplicates. See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Enhancement of E-Cadherin Function Increases Apoptotic Signaling via DR4 and DR5
(A) A schematic illustration for cell seeding configuration on coated substrate.
(B) C170 cells were dissociated, seeded for 2 hr on plates coated with E-cadherin (E) or poly-L-lysine (PLL), and stimulated by Apo2L/TRAIL for the indicated time. The cells were then lysed and analyzed by IB.
(C and D) C170 cells were dissociated, seeded on E-cadherin- or PLL-coated plates for 2 hr, and stimulated by Apo2L/TRAIL stimulation. Cells were assayed for caspase-8 and caspase-3 or -7 activity at 2 hr (C) and for cell death at 24 hr (D). Values are means ± SD of triplicates. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 E-Cadherin Enhances DISC Assembly
(A) PATU8902 cells were transfected with control siRNA (C) or siRNA against E-cadherin (E) for 48 hr, followed by Apo2L/TRAIL stimulation for the indicated time at 4°C. Cell lysates were subjected to IP with DR4 and DR5 antibodies. IP samples were analyzed by IB.
(B) PATU8902 cells were transfected with control or E-cadherin siRNA and treated with Apo2L/TRAIL (1 μg/ml) for 1 hr. Cell lysates were resolved by SEC, and caspase-8 activity present in column fractions was assayed. Protein standards were used to approximate molecular mass (kDa) of proteins in each fraction.
(C and D) PATU8902 cells were treated as in (B). High MW fractions (14–18) and low MW fractions (28–31) were subjected to IP with antibody against DR4 (C) or DR5 (D) and analyzed by IB. Blots for siControl and siE-cad in each antibody case were exposed onto a single film to enable direct comparison.
(E) PATU8902 cells were treated and resolved as in (B). High MW fractions (14–19) were analyzed by IB.
(F) PATU8902 cells were treated as in (B). High molecular weight fractions (14–19) were subjected to IP with DR4 and DR5 antibodies, and associated caspase-8 activity was measured.
(G) PATU8902 cells were transfected with control siRNA (C) or siRNA against E-cadherin (E) for 48 hr, followed by Apo2L/TRAIL stimulation for the indicated time. The cells were subjected to subcellular fractionation. The lipid raft/cytoskeleton fraction was analyzed by IB.
(H) NCI-H358 parental (P) and mesenchymal (M) cells were treated and analyzed as in (A).
(I) NCI-H358 parental (P) and mesenchymal (M) cells were treated and analyzed as in (G). See also Figure S4.
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7. Figure 5 E-Cadherin Physically Interacts with DR4 and DR5
(A) PATU8902 cells were left unstimulated (lanes 1 and 2) or treated with Apo2L/TRAIL for 30 min (lane 3). Cell lysates were subjected to IP with immunoglobulin G (IgG) (lane 1) or DR4 and DR5 antibodies (lanes 2 and 3). IP samples were analyzed by IB as indicated.
(B) Schematic illustration of the plate-based E-cadherin-DR4/DR5 binding assay.
(C) E-cadherin-Fc was added to protein A-precoated 96-well plates. Flag-DR4 and Flag-DR5 were added at increasing concentrations with or without Apo2L/TRAIL for 1 hr. The bound DR4 and DR5 were detected by antibody against Flag (M2), conjugated to HRP. Values are means ± SD of quadruplicates.
(D) The assay was performed and analyzed as in (C), using PBS or Ca2+-free PBS as indicated.
(E) The assay was performed and analyzed as in (C), using DR4 or the long (DR5L) or short (DR5S) splice variants.
(F and G) HEK293 cells were transiently cotransfected with expression vectors for DR5S (F) or DR5L (G), together with cytokine response modifier A (crmA), and E-cadherin domain deletion mutants as schematically illustrated (F). Cell lysates were subjected to IP with DR5 antibody and analyzed by IB. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
E-Cadherin Couples DR4 and DR5 to the Actin Cytoskeleton via α-Catenin
(A and B) We transfected C170 (A) and PATU8902 (B) cells with control siRNA or siRNAs against α-catenin for 48 hr, followed by Apo2L/TRAIL stimulation for 48 hr and analysis of cell death. Values are means ± SD of triplicates.
(C) C170 cells were transfected with control siRNA or siRNAs against α-catenin for 48 hr, followed by Apo2L/TRAIL stimulation. The cells were assayed for caspase-8 and caspase-3 or -7 activity at 2 hr. Values are means ± SD of triplicates.
(D) PATU8902 cells were transfected with control siRNA (C) or siRNA against α-catenin (αC) for 48 hr, followed by Apo2L/TRAIL stimulation for the indicated time at 4°C. Cell lysates were subjected to IP with DR4 and DR5 antibodies. IP samples were analyzed by IB.
(E) PATU8902 cells were transfected with control siRNA (C) or siRNA against α-catenin (αC) for 48 hr, followed by Apo2L/TRAIL stimulation for the indicated time. The cells were subjected to subcellular fractionation. The lipid raft/cytoskeleton fraction was analyzed by IB.
(F) PATU8902 cells were pretreated for 1 hr with EGTA (2 mM) and actin polymerization inhibitors latrunculin B (LacB) or cytochalasin D (CytoD) (2 μM). EGTA was washed away, and cells were replenished with fresh medium containing latrunculin B or cytochalasin D (2 μM) for an additional 1 hr, followed by Apo2L/TRAIL stimulation for 2 hr. Cells were assayed for caspase-8 and caspase-3 or -7 activity. Values are means ± SD of triplicates.
(G) PATU8902 cells were pretreated as in (F), followed by Apo2L/TRAIL stimulation for the indicated time at 4°C. Cell lysates were subjected to IP with DR4 and DR5 antibodies and analyzed by IB. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
E-Cadherin Expression in Epithelial Cancer Cell Lines Correlates with Sensitivity to Apo2L/TRAIL
(A–C) E-cadherin protein expression levels in panels of lung (A), colorectal (B), and pancreatic (C) cancer cell lines. Blue, Apo2L/TRAIL sensitive; red, Apo2L/TRAIL resistant.
(D) Summary of Apo2L/TRAIL IC50 values for the cells lines in (A)–(C).
(E) Model for E-cadherin regulation of apoptosis signaling via DR4 and DR5. Please see Discussion for a detailed description of the model.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions