Fig 2: Cell–cell clustering is driven by PVRL4-PVRL1 trans-interactions between individual cells.Clustering assays were performed with TL-HMECs expressing the following transgenes: (i) empty vector/control shRNA; (ii) PVRL4-CD8tm/control shRNA; (iii) PVRL4-CD8tm/anti-PVRL1 shRNA; and (iv) a 1:1 mixture of (iii) and (i).DOI: http://dx.doi.org/10.7554/eLife.00358.013

Fig 3: PVRL4-driven cell-to-cell attachment promotes anchorage-independence via integrin β4-associated signaling.(A) Cell surface-localized proteins interacting with HA/FLAG-tagged PVRL4, but not with HA/FLAG-tagged GFP, as determined by mass spectrometry. (B) TL-HMECs expressing HA/FLAG tagged PVRL4 or HA/FLAG-tagged GFP were detached from the adherent surface with the enzyme-free cell dissociation buffer and incubated in suspension for 1 hr. Immunoprecipitations were performed with HA beads, followed by Western blot with FLAG and integrin β4 antibody. (C) TL-HMECs expressing vector control, PVRL4, or myr-PI3K were stably transduced with the indicated shRNA constructs and integrin β4 levels were assayed by Western blot. (D) TL-HMECs from (C) were assayed for anchorage-independent colony formation. Colony numbers were normalized to the vector control sample. Assays were performed in triplicate (error bars ± SD). (E) TL-HMECs stably transduced with the indicated constructs were detached from the adherent surface with enzyme-free cell dissociation buffer and incubated in 0.5% methylcellulose in suspension for 6 hr or cultured on an adherent surface for 48 hr. Levels of pY416-SFK (Src family kinases), total SFK, and vinculin loading control were measured by Western blot. Band intensity was measured with ImageJ software. (F) PVRL4-expressing TL-HMEC cells transduced with control or anti-PVRL1 shRNA were incubated in suspension in the conditions indicated. Levels of pY416-SFK (Src family kinases) and tubulin loading control were measured by Western blot. Band intensity was measured with ImageJ software. (G) TL-HMECs stably expressing PVRL4 or control vector were assayed for anchorage-independent colony formation in the presence of PP2 or vehicle control. Colony numbers were normalized to the vector sample. Assays were performed in triplicate (error bars ± SD). (H) TL-HMECs expressing PVRL4 were stably transduced with the indicated shRNA constructs and assayed for anchorage-independent colony formation. Colony numbers were normalized to the vector control sample. Assays were performed in triplicate (error bars ± SD). (I) SHP-2 levels were assayed by Western blot in TL-HMEC lysates from (H).DOI: http://dx.doi.org/10.7554/eLife.00358.019

Fig 4: Depletion efficiency of individual anti-PVRL1 shRNAs.efficiency of shRNA-mediated PVRL1 mRNA depletion was measured by RT-qPCR. PVRL1 transcript abundance was normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD).DOI: http://dx.doi.org/10.7554/eLife.00358.008

Fig 5: PVRL4 facilitates cell-to-cell attachment, inhibition of which suppresses anchorage-independence.(A) and (B) PVRL4 promotes cell clustering of TL-HMECs. Cells were dissociated off the tissue culture surface with trypsin-free cell dissociation buffer and kept in suspension for 1 hr. Small (3–5 cells) and large (>5 cells) cell clusters per field of view were counted, n = 3 (error bars ± SD). (C) GFP-labeled PVRL4-expressing TL-HMECs were allowed to aggregate with dsRed-labeled cells expressing either a PVRL1-targeting shRNA or a control shRNA. Representative phase-contrast and fluorescent images (red and green channels superimposed) are shown. (D) PVRL4 was co-expressed with the indicated shRNAs and anchorage-independent colony formation in TL-HMECs was assayed. Values were normalized to an empty vector-transduced sample. Assays were performed in triplicate (error bars ± SD). (E) PVRL4-expressing TL-HMECs were assayed for clustering in the presence of the indicated antibodies or isotype controls. Cell clusters were quantified as before. (F) Anchorage-independent growth induced by PVRL4 or an shRNA against PTEN was assayed in the presence of PVRL4-targeting antibody or control IgG. Colony numbers were normalized to the control sample. Anchorage-independent colony formation assays were performed in triplicate (error bars ± SD).DOI: http://dx.doi.org/10.7554/eLife.00358.005