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. 2013 Apr 30;2:e00358. doi: 10.7554/eLife.00358

Figure 6. PVRL4 is amplified in breast cancer and is essential for the transformed phenotype of cancer cells.

(A) A view from the integrated Genome Viewer program showing focal amplification of the PVRL4 locus in SUM190 cells. The degree of amplification is denoted by the intensity of the color. (B) PVRL4 mRNA was stably depleted from SUM190 cells by four independent shRNAs. Transcript levels were measured by RT-qPCR and normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD). PVRL4-depleted and control cells were assayed for clonogenic survival and anchorage-independent colony formation. Assays were performed in triplicate (error bars ± SD). All values were normalized to the uninfected control sample. ECM: extracellular matrix. (C) The PVRL4-CD8 chimeric construct was used to rescue the defect in clonogenic survival observed with RNAi-mediated PVRL4 depletion. Assays were performed in triplicate (error bars ± SD). Colony numbers were normalized to the control shRNA sample. (D) Expression levels of endogenous and chimeric proteins were verified by Western blot.

DOI: http://dx.doi.org/10.7554/eLife.00358.021

Figure 6.

Figure 6—figure supplement 1. PVRL4 induces clustering of SUM190 cells which is blocked by antibodies against PVRL4.

Figure 6—figure supplement 1.

SUM190 cells were assayed for cell–cell clustering in the presence of the indicated antibodies. Four-cell clusters and clusters with more than four cells from representative images were scored separately.
DOI: http://dx.doi.org/10.7554/eLife.00358.022
Figure 6—figure supplement 2. PVRL4 induces clustering of SUM190 cells which is blocked by RNAi against PVRL4.

Figure 6—figure supplement 2.

SUM190 cells were assayed for cell–cell clustering in the presence of the indicated shRNAs. Four-cell clusters and clusters with more than four cells from representative images were scored separately.
DOI: http://dx.doi.org/10.7554/eLife.00358.023
Figure 6—figure supplement 3. PVRL4 induces attachment of SUM190 cells to microvascular endothelial cells which is blocked by antibodies against PVRL4.

Figure 6—figure supplement 3.

SUM190 cells (GFP) were assayed for heterotypic clustering with HMVEC-L cells (dsRed) in the presence of the indicated antibodies. Clusters with at least three cells incorporating both green and red cells from representative images were counted.
DOI: http://dx.doi.org/10.7554/eLife.00358.024
Figure 6—figure supplement 4. PVRL4 induces attachment of SUM190 cells to microvascular endothelial cells which is blocked by RNAi against PVRL4.

Figure 6—figure supplement 4.

SUM190 cells (GFP) were assayed for heterotypic clustering with HMVEC-L cells (dsRed) in the presence of the indicated shRNAs. Clusters with at least three cells incorporating both green and red cells from representative images were counted.
DOI: http://dx.doi.org/10.7554/eLife.00358.025
Figure 6—figure supplement 5. Stable depletion of PVRL4 transcript in BT-474 and Sk-BR-3 cell lines.

Figure 6—figure supplement 5.

PVRL4 transcript levels were measured by RT-qPCR and normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD).
DOI: http://dx.doi.org/10.7554/eLife.00358.026
Figure 6—figure supplement 6. PVRL4 depletion affects clonogenic growth of BT-474 and Sk-BR-3 cell lines.

Figure 6—figure supplement 6.

The clonogenic potential of the indicated cell lines in the presence of control or PVRL4 shRNA constructs was assessed. Assays were performed in triplicate (error bars ± SD). Colony numbers were normalized to the control shRNA sample.
DOI: http://dx.doi.org/10.7554/eLife.00358.027
Figure 6—figure supplement 7. PVRL4 depletion affects anchorage-independent growth of BT-474 and Sk-BR-3 cell lines.

Figure 6—figure supplement 7.

Cell line growth in methylcellulose-containing media on an ultra-low attachment surface was assessed in the presence of control or PVRL4 shRNA constructs. Representative images are shown.
DOI: http://dx.doi.org/10.7554/eLife.00358.028