Overview on amplified chromosomal regions.

<p>Chromosome regions that overlap with gained chromosome regions of TCGA glioblastoma samples are indicated in bold. Likewise, genes that were used for FISH analysis are indicated in bold. Examples of glioblastoma-amplified genes were included for chromosomal regions amplified after 5 d of differentiation. Start and end point were according to NCBI36/HG18.</p

FISH and immunofluorescence analysis.

<p>FISH with <i>GINS2</i> specific BAC (pink) and simultaneous immunofluorescence staining with GFAP (green) revealed <i>GINS2</i> amplification in cells with beginning GFAP expression after 5 d of differentiation (A). FISH with <i>CDK4</i> specific BAC (pink) and immunofluorescence staining with GFAP revealed <i>CDK4</i> amplification in cells with beginning GFAP expression after 2 d of differentiation (B). FISH with <i>CDK4</i> specific BAC (pink) and immunofluorescence staining with Tubulin-ß-III-chain (green) revealed <i>CDK4</i> amplification in cells with beginning Tubulin-ß-III-chain expression after 11 d of differentiation (C and D). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm.</p

Correlation of gene content to chromosome region.

<p>The correlation is shown for human chromosome 16 with several amplified and one deleted region in NHNP cells after 5 d differentiation. The log₂ ratio profile of the 10× window averaged data is presented at 25 kb resolution (A). Fused lasso analysis is performed using the same data points (B). GC repeats, gene content and banding pattern is shown as indicated by Ensembl genome browser (C). Both the log₂ ratio profile and the fused lasso analysis indicate an overlap between amplifications and regions with high GC and gene content and an overlap between deletion and regions with low GC and gene content.</p

Whole chromosome plots.

<p>A genome-wide view of the 10× window-averaged data at 25 kb resolution is displayed for NHNP cells at day 0, day 1, day 2 and day 5 during differentiation.</p

FISH analysis of amplified loci.

<p>For each FISH analysis, a BAC or cosmid clone containing the indicated gene was Cy3-labelled (pink) and hybridized against fixed NHNP cells that were differentiated for either 2, 5 or 7 days. Amplifications are shown for <i>C1QL1</i> RP11-113A24 (5 days), <i>SOX13</i> RP11876H8 (5 days), <i>HDGF</i> RP11-66D17 (5 days), <i>CYP27B1</i> cosmid (2 days), <i>GINS2</i> RP11-118F19 (2 days), <i>CDK4</i> RP11-571M6 (2 days), <i>TP53</i> RP11-1081A10 (7days), <i>DIABLO</i> RP11-568C23 (7 days). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm. Notably, the degree of amplification various within each analysis due to the high heterogeneity of the amplifications in each cell population.</p

Detailed gene amplification analysis on human chromosome 16 and 12.

<p>Representative sections of log₂ ratio profiles for undifferentiated (0 d) NHNP cells and cells that were differentiated for 2 and 5 days. Base count is given on the x-axis and log₂ ratio on the y-axis for chromosome 16q24.1 (A) and 12q14.1 (C). Chromosomal localization of BAC probes used for FISH were indicated at the bottom of figures C and D. A <i>GINS2</i> specific BAC probe that was labeled in pink and a <i>CDH13</i> specific BAC probe that was labeled in green were hybridized simultaneously against fixed NHNP cells that were differentiated for 2 days. <i>GINS2</i> amplification is indicated as pink speckled fluorescence signals whereas the neighboring <i>CDH13</i> gene shows only single copy fluorescence signals (Bi). Neighboring cells with and without <i>GINS2</i> amplification are shown in Figure Bii. A <i>CDK4</i> specific BAC probe that was labeled in pink and a <i>XRCC6BP1/KUB3</i> specific BAC that was labeled in green were hybridized simultaneously against NHNP cells that were differentiated for 2 days. <i>CDK4</i> and <i>KUB3</i> amplifications were detectable as cluster of pink and green speckled fluorescence signals. <i>CDK4</i> specific signals spread over a more extended area than the <i>KUB3</i> specific signals (D). Nuclei were counterstained with DAPI (B). Size calibration bar = 5 µm.</p