* = < 0.05 1< 0.05 RPM AD. Open in a separate window Figure 4 Quantitative real-time PCR of genes of interest II and secretion. exhibited a similar behavior overall with some differences in detail concerning gene and protein expression as well as the speed of aggregation . The MCS are of great interest for cancer research, because they provide the possibility to study cancer cells (Partek analyses) methods. 2. Results and Discussion 2.1. Spheroid Formation Phase contrast microscopy revealed that the static control UCLA RO82-W-1 cells grew as a normal monolayer, when the cell culture flasks were placed next to the RPM in the incubator (Figure 1a). However, after a 24 h-exposure of UCLA RO82-W-1 follicular thyroid cancer cells to the RPM, two types of growth were observed: irrespectively of the culture dish used, one part of the cells grew as 3D aggregates, the other part as a two-dimensional monolayer (Figure 1b,c). Open in a separate window Figure 1 Phase Cryptotanshinone contrast microscopic pictures of UCLA RO82-W-1 1gene expression analysis. Rhodamine-phalloidin staining of UCLA RO82-W-1 cells exposed to SDF-5 1(a) and altered gravity on the RPM; (b) for 24 h. White arrows designate stress fibers accumulating at the cell borders, the yellow arrow shows lamellipodia and filopodia, the orange one microvilli. Quantitative rtPCR analyses of the gene expression in 1(a); (b); (c); (d); (e); (f); (g); and (h) gene expression levels. * = < 0.05 1< 0.05 RPM AD. Open in a separate window Figure 4 Quantitative real-time PCR of genes of interest II and secretion. After 24 h, 1(a); (b); (c); (d); (f); (g); and (h) gene expression levels. In addition, secretion Cryptotanshinone into the culture medium was determined (e) * = < 0.05 1< 0.05 RPM AD. Open in a separate window Open in a separate window Figure 5 Quantitative real-time PCR of genes of interest III. After 24 h, 1g-control, RPM-adherent (AD) and RPM-multicellular spheroids (MCS) were analyzed for their MMP3 (a); CAV1 (b); TBFB1 (c); PAI1 (d); MMP9 (e); CAV2 (f); TGFBR1 (g) and RHOA (h) gene expression levels. * = < 0.05 1< 0.05 RPM AD. Open in a separate window Figure 6 Mutual regulation network at gene expression level. and cytoskeletal proteins. The gene expression of and was reduced in MCS and AD cells (Figure 3a,e) compared with 1and was downregulated in MCS (Figure 3b,f). Furthermore, the EZR mRNA was clearly decreased in RPM AD samples and further downregulated in MCS compared with 1gene expression significantly in both groupsAD and MCS (Figure 3d), whereas decreased in AD, Cryptotanshinone but was elevated in MCS (Figure 3g). There was a tendency of upregulation in AD cells and of a downregulation in MCS (Figure 3h). Taking together the results shown in Figure 3 clearly shows that the expression of the examined genes related to the cytoskeleton was downregulated during spheroid formation early within 24 h 2.3. Three-Dimensional Growth Signaling Pathways Nothing is known about the role of typical angiogenic factors in the early phases of MCS formation. Here we detected a 2.5-fold increase in the gene expression in MCS samples (Figure 4a) and a 5-fold increase in VEGF protein released in the supernatant (Figure 4e). We could also find an upregulated gene expression of in MCS samples of UCLA RO82-W-1 cells (Figure 4b). The (gene expression remained unchanged (Figure 4c). Focusing on the protein kinase C (PKC) pathway, which is involved in cell proliferation and is affected during spheroid formation , we could measure a clear reduction of mRNA after RPM-exposure in AD and MCS samples compared with corresponding static 1mRNA was downregulated (Figure 4d). Monocyte Chemoattractant Protein-1 (MCP-1) had been identified to play an important role in 3D formation under conditions of weightlessness . The gene expression was downregulated in MCS (Figure 4h), but remained unchanged in AD cells. Hence Figure 4 shows that two of the angiogenic-related genes (mRNA in RPM AD cells and MCS cultures, as well as spaceflight AD cells and MCS cultures . Interestingly, the mRNA was increased in the RPM and in Space cultures of the Shenzhou-8 Space Mission , which is comparable to Cryptotanshinone the 24 h-result in UCLA RO82-W-1 RPM samples. 2.4. Factors Regulating the Amount of Extracellular.