Effects of EGFR activation/blockade on spheroid volume.

<p>Cells were seeded at a density of 300,000 cells/well into poly-HEMA coated 6-well plates and treated with the indicated agents. After 72h, the diameters of at least 15 spheroids were measured and spheroid volumes calculated relative to the untreated spheroids. Bars represent mean values ± SD of four independent experiments. For the cell line SCC-9 the y-axis was adapted due to the EGF-induced strong increase in spheroid volumes.</p

CTX affects proliferation of spheroids in sensitive cell lines.

<p>MTT assays were performed in 96-well plates with cells treated with EGFR stimulating or blocking reagents in monolayer (M) or forced suspension (FS) culture. Bars represent mean percentages ± SD of four independent experiments. Significant changes compared to control are marked with an asterisk.</p

EGFR signalling regulates clonogenic survival of HNSCC cells derived from forced suspension cultures.

<p>Cells were cultured as monolayer (M) or in forced suspension (FS) in the absence or presence of EGF, AREG or CTX. 72h later, cells were harvested, disaggregated to single cells and subjected to clonogenic survival analysis. Bars represent the surviving fractions (SF) ± SD from three independent experiments. Significant changes compared to control are marked with an asterisk.</p

Autocrine EGFR signalling is maintained in spheroid culture of HNSCC cell lines.

<p>Cells were cultured as monolayer (M) or spheroids (FS) for 24h, 48h or 72h in serum-free medium. The expression levels of pERK1/2 (42/44 kDa) and vinculin (internal loading control; 124 kDa) were analysed by immunoblotting. The values of the quantitative analysis are depicted as pERK1/2 protein levels relative to vinculin expression levels.</p

Activation of EGFR protects HNSCC cells against anoikis.

<p>Cells were seeded at a density of 300,000 cells/well in non-coated or poly-HEMA coated 6-well plates and were immediately treated with either EGFR ligands or blocking reagents. After 72h, cells from monolayer (M) and forced suspension (FS) cultures were harvested and the apoptotic fraction was determined by PI staining and subsequent flow cytometry. Bars represent the mean percentages of apoptotic cells ± SD of at least three independent experiments. Significant changes compared to control are marked with an asterisk.</p

Spheroid generation is cell line dependent.

<p>(A) 2,500 cells were seeded into 96-well plates (poly-HEMA coated). After 96h, total number of spheroids per well were counted. (B) 2 ×10⁴cells/well were plated into 12-well culture plates and at indicated times cells were counted. The data points shown in (A) and (B) represent the mean values ± standard deviation (SD) of three independent experiments. (C) Cells were lysed, and protein samples were subjected to western blot analysis with specific antibodies against EGFR. GAPDH was used as a loading control.</p

<i>NIPBL-AS1</i> does not influence <i>NIPBL</i> transcription.

<p>A) Overview of the genomic position of <i>NIPBL</i> and <i>NIPBL-AS1</i> genes. Strand-specific read coverage of RNA-sequencing data (positive in green; negative in red) from HEK293T cells shows the transcription of <i>NIPBL-AS1</i> antisense to <i>NIPBL</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref001" target="_blank">1</a>]. CTCF binding sites in HEK293 cells (ENCODE hg18) are shown. Primers used in the transcript analysis are indicated as green bars. (B-C) Transcript levels of (B) <i>NIPBL-AS1</i> and (C) <i>NIPBL</i> after antisense oligonucleotide knockdown (ASO2, ASO3) of <i>NIPBL-AS1</i> in HEK293T cells. ASO C was used as control. Transcript levels were normalized against the control sample (ASO C) and the housekeeping <i>SNAPIN</i> using the ΔΔCt method (mean n = 3, error bars +/- s.d., p-values determined with t-Test).</p

Implications for CdLS.

<p>A) Transcript levels of the genes <i>BBX</i>, <i>GLCCI1</i> and <i>ZNF695</i> that were described as dysregulated genes in CdLS [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref020" target="_blank">20</a>] and previously confirmed as NIPBL-dependent genes with NIPBL binding sites at the promoter [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref008" target="_blank">8</a>] were analysed in the different enhancer deletion clones D1 and D2 (mean n = 5 for D1 and n = 4 for D2, error bars +/- s.d., p-values determined with t-Test, the transcript levels of the individual clones are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.s007" target="_blank">S7 Fig</a>). B) Average transcript levels of <i>NIPBL</i> and <i>NIPBL-AS1</i> in lymphoblastoid cell lines (LCLs) derived from CdLS patients and controls. The details of the four LCL controls and three CdLS LCLs as well as the individual transcript levels are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.s008" target="_blank">S8 Fig</a> and in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref008" target="_blank">8</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref020" target="_blank">20</a>]. Two primer pairs for <i>NIPBL</i> and one for <i>NIPBL-AS1</i> were used. Transcript levels were normalized against the housekeeping gene <i>NADH</i> (mean n = 4 for control LCLs and n = 3 for CdLS LCLs, error bars +/- s.d., p-values determined with t-Test).</p

Interactions of <i>NIPBL</i> and <i>NIPBL-AS1</i> with a potential distal enhancer.

<p>A) Long-range chromosomal interactions of the <i>NIPBL</i> and <i>NIPBL-AS1</i> promoter detected by chromosome conformation capture (3C-seq) in HEK293T cells using an ApoI digest. The positions of the different viewpoints used are marked in yellow. Three different viewpoints at the promoter (VP4, blue track) and the candidate enhancers regions R1 (VP5, green track) and R2 (R2—VP6, red track) were used. B) CTCF ChIP sequencing track from HEK293 cells (ENCODE). The orientations of the CTCF motifs as determined with JASPAR are shown below the track (red triangle–forward orientation, green triangle–reverse orientation). The CTCF sites involved in the promoter-enhancer interaction are indicated with yellow triangles above the track. C) DNAse clusters as well as histone modification profiles—H2A.z, H3K4me1, H3K4me2 and H3K4me3—of six different cell lines (G312878, K562, HeLa-S3, HEMEC, HSMM and HUVEC, available from ENCODE) are displayed as density graph. Black represents areas with the highest enrichment of the signals.</p