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Fifty-percent MSC-CM resulted in 9.6 and 21.0-fold increase in sphere formation after 5 days for HMEC and MCF-7 cells (Figure 3A, 3B and 3D).
HMEC cells plated with MSC demonstrated a dose-dependent increase in mammosphere formation, with a nearly two-fold increase in sphere formation in the presence of 10% MSC (Figure 1B, p<0.01).
There was a statistically significant increase in sphere forming frequency with 1 µM (p = 0.0004, 2 days) and 2.5 µM 4-OHT (p = 0.014, 2 days; p = 0.021, 6 days) treatments (Figure 6B).
A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3).
We observed a substantial (approximately 8-fold) increase in sphere forming potential of cells derived from the Ptc1Lox/Lox NestinCre neocortex (2.154±0.267%) compared to Ptc1Lox/Lox neocortex (0.27±0.047%, p<0.0001,) (Fig. 2A) indicating that Hh pathway activation in vivo can regulate the total number of stem and progenitor cells.
Interestingly, a 4.6-fold increase in sphere forming potential was observed in Ptc1Lox/Lox RbpjLox/Lox NestinCre neocortex compared to RbpjLox/Lox NestinCre neocortex (p = 0.0427) suggesting that Hh signaling can compensate for loss of Notch signaling to support the sphere forming properties of neocortical progenitors (Fig. 2A).
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MCF7Podxl cells (Additional file 3C) exhibited a 30% increase in sphere-forming efficiency.
Consistent with the EMT promoting self-renewal gene expression, we found a significant increase in sphere-forming activity in TGF β1-treated H441 and primary NSCLA cells.
These results are in line with a report demonstrating that CD133 expression is increased in spheres but not in every analyzed sphere derived from NB samples and cell lines.
In addition, the expression of mRNA of the transcription factors involved in EMT, such as Twist, Snail, and mesenchymal makers, such as Vimetin and N-cadherin were also increased in sphere-derived cells compared to the parental cells.
29, 45 Detailed analyses of cell lines derived from other types of pediatric brain tumors (i.e., ependymoma, medulloblastoma, glioma, and primitive neuroectodermal tumors) revealed that the number of nestin-positive CSCs is increased in spheres exhibiting a CSC phenotype based on functional assays.
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