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