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Switching from Epo to SCF induced a marked increase in Kit level (Figure 3A).
Strikingly, inhibition of EpoR signaling in AG490-treated cells was accompanied by an increase in Kit expression as compared to untreated cells.
As illustrated in Figure 5B, expression of MT-LynY397F detected by immunoblotting with the MT antibody was associated with an increase in Kit level in the Epo-cultured cells.
The overall increase in SSC number (SSC concentration × cell increase) in Kit+ and Kit− cells was 17.5 and 16.0-fold, respectively, and the difference was not statistically significant (Figure 3H).
Indeed, TOC-resistant sublines demonstrated up to a four-fold increase in KIT receptor expression compared to the parental, treatment naïve C2 cells.
To determine if this increase in c-kit transcript led to a subsequent increase in KIT receptor expression, flow cytometry was performed.
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To determine whether an increase in the KIT copy number contributes to its over-expression, we undertook its quantification studies.
In the current study, analysis of c-kit mRNA expression by real-time quantitative PCR demonstrated a significant increase in c-kit expression in the TOC-resistant C2 sublines compared to the treatment-naïve parental C2 cells.
After 2 d in serum-free culture (−BMP4) there was a minimal increase in CD34+/c-Kit+ cells although the ratio of CD34+/c-Kithigh and CD34+/c-Kitlow cells was similar to day 0. In contrast, addition of BMP4 (+BMP4) resulted in a greater expansion in the CD34+/c-Kitlow population (from 3·2%too 11·6%) compared to day 0 and serum-free controls.
Differently, despite a discrete concentration of VEGF and EGF in the PL preparations used in this study, we have not observed a preferential commitment to the endothelial lineage but to an increase in c-kit gene expression levels, which is normally not expressed by ADMSCs after isolation [ 8].
We confirmed that Fgf9 treatment increases Nanos2 expression in Kit+ spermatogonia, and found that this effect also required Nodal activity, as it was abolished by SB431542 treatment.
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