Methylation status of CpG sites in partially methylated samples (i–iii) were determined by bisulfite sequencing ()

<p><b>Copyright information:</b></p><p>Taken from "Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorectal cancer patients"</p><p>Nucleic Acids Research 2005;33(5):e46-e46.</p><p>Published online 10 Mar 2005</p><p>PMCID:PMC1064143.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Probes 2–4 cover the 10 CpG sites in these samples. () The methylation levels detected by the oligonucleotide microarray were compared with those derived from bisulfite sequencing. The methylation levels by bisulfite sequencing was determined by dividing the total number of methylated CpG sites analyzed by the number of CpG sites at that locus and multiplying by 100, whereas the percentage of methylation levels analyzed by microarray analysis was determined by the use of standard curves derived from the aforementioned calibration controls as shown in . The average signal intensity was taken from two spots on four slides proceeded in parallel. The error bars indicate SD

Tumor morphologies and metastatic potentials of the prostate-derived cell lines.

<p>Tumor morphologies and metastatic potentials of the prostate-derived cell lines.</p

Enlargement of GLA due to slow cell proliferation and inter-gla fusion in the 3d stemness-inducing nanoenvironment.

<p>(A) Representative photomicrographs of PC-3 cells after reaching confluent. Cells were cultured in the 2D condition. Cellular morphologies at day 4, 5, and 7 were shown. Arrowheads indicate GLA on the 2D monolayer cells. Scale bar, 100 μm. (B) Representative photomicrographs of PC-3 cells in the 3D culture condition. Cellular morphologies at day 11 and 14 were shown. Scale bar, 100 μm. (C) Growth curves of PC-3 cells cultured in 2D serum-contained and 3D stem cell medium conditions. Cells were cultured in a 96-well plate. **P < 0.01 (2D serum vs 3D stem), n = 3. (D) Growth curves of PC-3 cells cultured in 2D serum-contained and 2D stem cell medium conditions. *P < 0.05 (2D stem vs 2D serum), n = 3. (E) Growth curves of PC-3 cells cultured in 3D serum-contained and 3D stem cell medium conditions. *P < 0.05 (3D stem vs 3D serum), n = 3. **P < 0.01 (3D stem vs 3D serum), n = 3. (F-H) Viabilities of PC-3 cells cultured in 2D or 3D conditions in serum-contained or stem cell media. Same data with different vertical axis values were shown between F and H and between G and I. (F, H) P < 0.05 (2D serum vs 2D stem), n = 3. (G, I) *P < 0.05 (vs day 0), n = 3. **P < 0.01 (vs day 0), n = 3. 3D serum d0 vs d7, P = 0.028. 3D serum d0 vs d11, P = 0.0052. 3D serum d0 vs d14, P = 0.0012. 3D stem d0 vs d7, P = 0.0138. 3D stem d0 vs d11, P = 0.0007. 3D stem d0 vs d14, P = 0.0004.</p

Classification of the morphologies of cellular aggregations.

<p>Classification of the morphologies of cellular aggregations.</p

Gene expression switching of Epithelial-Splicing Regulatory Proteins (ESRPs), CD44 variant, and stem cell markers depending on cell culture nanoEnvironments.

<p>(A) Representative morphologies of PC-3 cells cultured in the 4 different conditions. Cells were cultured in 10% serum-containing F12K medium or mTeSR1 stem-cell medium on 2D plates or 3D NCPs. Arrows indicate projections of cells. Scale bars, 100 μm. (B) Schematic structures of <i>CD44</i> gene, <i>CD44 variant 8–10</i> (<i>CD44v8-10</i>) and <i>CD44 standard</i> (<i>CD44s</i>). Blue and gray rectangles represent standard exons (exon 1 to 10) and variant exons (V1 to V10), respectively. The red primer pair is for all variants and the CD44s. The green primer pair is for CD44v containing exon V9. The blue primer pair is for CD44s only. (C) Agarose gel electrophoresis analysis of RT-PCR amplicons of CD44v and CD44s. An arrow indicates <i>CD44v8-10</i> amplicon. An arrowhead indicates <i>CD44s</i> amplicon. M1k, a 1 kbp DNA ladder marker. M100, a 100 bp DNA ladder marker. <i>ACTB</i>, β-actin mRNA as an internal control. (D) qRT-PCR analysis of stem-cell-related and epithelial-splicing regulatory genes. The mRNA expression levels of <i>CD44s</i>, <i>CD44v</i>, <i>ECAD/CDH1</i>, <i>ESRP1</i>, <i>ESRP2</i>, and <i>CD133</i> were examined. Relative mRNA expression levels versus those of <i>GAPDH</i> are shown. n = 3. (E) Flow cytometry analysis of CD44v9. PC-3 cells were cultured in serum-containing medium and passage number 1, 7, and 13 were examined by flow cytometry. An anti-prostate-specific antigen (PSA) antibody was used as a negative control. Serum promoted differentiation of the cells and reduced stemness.</p

Positivity rates of markers of cancer-stem, neuroendocrine, mesenchymal, and endothelial cells.

<p>Positivity rates of markers of cancer-stem, neuroendocrine, mesenchymal, and endothelial cells.</p

The difference in tumorigenicity and metastatic potentials between spheroid-forming and GLA-forming adenocarcinomas.

<p>(A) In vivo tumorigenicity of PC-3 (GLA-forming), DU-145 (spheroid-forming), LNCaP (other type of aggregates), and RWPE-1 (monolayer sheet-forming) cells. Each cell line was pre-cultured under the 2D conditions and transplanted to SCID mice. The PC-3 cells were pre-cultured in 2D or 3D conditions. The photographs on day 27, 52, 72, and 79 after the transplantation were shown. Arrows indicate tumors. Margins of tumors were traced with dotted lines. Tumor sizes were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191109#pone.0191109.t002" target="_blank">Table 2</a>. (B) Tumor growth of each cell line. (C) Primary tumor and lymph node metastasis of the PC-3 cells on day 52. The primary tumor was shown with dotted line. Metastasis was seen in the regional lymph nodes of the prostate (white arrows) and an axilla lymph node (arrow).</p

Subcellular localization of EpCAM, E-cadherin, and vimentin in the CSC-like 3D aggregates of neuroendocrine adenocarcinoma cells.

<p>PC-3 cells were cultured in 3D stem (A, C, E) and in 3D serum (B, D, F) conditions. Immunohistochemistry was carried out of EpCAM (A, B), E-cadherin (C, D) and Vimentin (E, F). DNA was stained with DAPI. Scale bars, 100 μm. Arrow indicates acinus-like structures. Arrowhead indicates duct-like structures.</p

Quantification of size and hypoxia level of GLAs in serum-stimulated and stemness-induction conditions.

<p>(A) The grape-like aggregation (GLA) of PC-3 cells in the serum-contained or stem-cell medium. Scale bar, 100 μm. (B) Time-lapse imaging of maturation of GLA of PC-3 cells. Fusion of small GLAs was seen to form larger GLAs in the stem cell induction condition. Arrowheads indicate pre-fusion aggregates. Scale bar, 100 μm. (C) Numbers of cellular aggregations per field. A cellular aggregation sized more than 100 μm² diameters was defined as a GLA. A well of a 96-well plate was sectionized to 9 fields, and the area of 1 field is 2,632 μm². n = 4. Biological replicates. (D) The average area of GLAs. n = 3. Biological replicates. (E) Hypoxia levels of the aggregations. n = 3. Biological replicates. (F) Hypoxia imaging by using a hypoxia probe. Scale bars, 200 μm. (G) Scatter plot analysis of areas and hypoxic levels of the GLAs. GLAs in random 4 fields were analyzed. Approximate straight lines were shown. Left, n = 1338. Right, n = 27. (H) The difference in sizes of GLAs. Gray, 100 to 500 μm²; orange, 500 to 5000 μm². Red, > 5000 μm².</p

Representative morphologies of the various types of cell aggregates formed on NanoCulture Plates (NCP).

<p>(A) Grape-like aggregation (GLA) of PC-3 cells formed in F12K medium with 10% FBS. (B) Spheroids of CAL 27 cells formed in DMEM with 10% FBS. (C) Cell aggregation of FaDu cells formed in DMEM with 10% FBS. (D) Epithelial monolayer sheet of RT7 cells formed in KGM2 medium. Scale bar, 100 μm. The morphological classification of 67 cell lines on NCP is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191109#pone.0191109.t001" target="_blank">Table 1</a>.</p