Molecular Recognition of DNA Double Helix by Abhijit Saha

Author:Abhijit Saha
Language: eng
Format: epub, pdf
Publisher: Springer Singapore, Singapore

3.2.2 Effect of HpβCD in MEF Treated by 1

Next, we thought if the barrier of solubility could be overcome by employing HpβCD in cell culture. Since the endogenous mRNA levels of the target genes can be inferred as a biological readout of the effectors, quantitative real-time polymerase chain reaction (qRT-PCR) was used to monitor the cellular uptake of PIPs. As compound 1 can induce Oct-3/4 pathway genes, thus those selected genes were used. Relying on the fact, we carried out screening studies to evaluate the effect of CD (5 or 50 mM) on the endogenous expression of the core pluripotency genes. While 1 notably induced the endogenous expression of the core pluripotency genes as reported before,10 CD alone did not influence any of the genes under study (Fig. 3.3). Treatment of 1 dissolved in either 5 or 50 mM of HpβCD showed only a marginal (a maximum of about 1.5-fold) increase in the endogenous expression of the core pluripotency genes (Oct-3/4, Nanog, Cdh1, Rex1, Sox2, and Dppa4) (Fig. 3.3). Statistical analyses verified that this marginal increase observed with the treatment of 1 with CD (5 or 50 mM) is not significant. Therefore, CD could improve the solubility of 1 under in vitro conditions but as a vehicle it could not significantly enhance its biological activity. Thus, an alternative strategy is required to improve the biological efficacy of 1.

Fig. 3.3Effect of HpβCD (5 and 50 mM) on 1 on the endogenous expression of pluripotency genes. Expression levels of a Oct3/4, b Nanog, c Rex1, d Cdh1, e Sox2, and f Dppa4 were determined by treating MEF with 100 nM of 1 to each sample, and with 0.1% DMSO, 5 and 50 mM of CD as a control experiment. Each bar represents the mean ± SD from 12-well plates

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