12 research outputs found

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

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    <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

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

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    <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

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

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    <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

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

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    <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 μm<sup>2</sup> 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 μm<sup>2</sup>. 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 μm<sup>2</sup>; orange, 500 to 5000 μm<sup>2</sup>. Red, > 5000 μm<sup>2</sup>.</p

    Secretion of EpCAM-exosomes, CD9-exosomes, and HSP90 by 3D aggregates of multipotent neuroendocrine adenocarcinoma cells.

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    <p>(A) TEM of exosomes secreted by PC-3 cells. Scale bar, 100 nm. (B, C) Cellular (B) and exosome (C) protein concentrations per million cells. PC-3 cells were pre-cultured the four different conditions and further cultured in serum-free medium for 2 days to prepare exosome and non-exosome fractions. (D) Western blotting analysis of EpCAM, CD9, and HSP90α in cellular, exosome, and non-exosome fractions. Each protein sample per 3 x 10<sup>5</sup> cells was used for analysis of exosome, per 1 x 10<sup>5</sup> cells was used for analysis of non-exosome fraction, and per 2 x 10<sup>4</sup> cells was used for analysis of cell lysates. (E) Western blotting analysis of E-cadherin and Vimentin. Each protein sample per 2 x 10<sup>4</sup> cells was loaded. β-actin and GAPDH were analyzed as loading controls. (F) Immunocytochemistry of EpCAM, vimentin, chromogranin A (CHGA), synaptophysin (SYP), and CD34 in the 2D culture conditions. Scale bar, 50 μm. Percentages of positive cells were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191109#pone.0191109.t004" target="_blank">Table 4</a>. Photomicrographs taken at a 20 x magnification is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191109#pone.0191109.s001" target="_blank">S1 Fig</a>.</p

    Gene expression profiling of CSC markers in the 2D and 3D culture nanoenvironments.

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    <p>PC-3 cells were cultured in four different conditions designated: 2D, serum; 2D, stem; 3D, serum; 3D, stem conditions. (A) Clustergram and dendrogram analysis of stem-cell-related genes. (B) Building block analysis of stem-cell-related genes. (C) Scatter plot analysis. Genes were plotted according to the mRNA expression levels in the 3D stem-cell condition (ordinate) and the 2D serum-containing condition (abscissa). <sup>#</sup>These gene’s average threshold cycle is relatively high (> 30) in any sample, and is reasonably low in the other sample (< 30).</p
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