Iation and functions. It has been reported that the expression of ER is higher and constant in the course of earlier stages of adipogenesis Saracatinib Description compared to ER, whose expression and continuous through earlier stages of adipogenesis in comparison to ER, whose expression dedecreases through adipocyte differentiation, suggesting an association between the exprescreases during adipocyte differentiation, suggesting an association among the expression sion of ERs and adipogenesis progression . Therefore, we analyzed the impact of Sof ERs and adipogenesis progression . Consequently, we analyzed the effect of S-equol equol on each ERs by real-time qRT-PCR (Figure(Figurepreviously reported, ER mRNA on both ERs by real-time qRT-PCR assays assays six). As 6). As previously reported, ER mRNA levels remained constant in the course of the differentiation method in 5-Azacytidine Purity manage cells; in conlevels remained constant during the differentiation method in manage cells; in contrast, trast, were had been lowered by about 48 and 62 in cells treated with S-equol on daysand 7, they they lowered by about 48 and 62 in cells treated with S-equol on days three 3 and 7, respectively, with respect control cells (Figure 6A).6A). Regarding ER, its expression respectively, with respect to to control cells (Figure Concerning ER, its expression was was decreased by about 59 on 7 in handle cells;cells; treatment with S-equol did not have an effect on decreased by about 59 on day day 7 in control treatment with S-equol did not affect ER ER mRNA levels on3, but3, but it produced a 43 areduction on day 7 (Figure 6B). These mRNA levels on day day it only only made 43 reduction on day 7 (Figure 6B). These data indicated that S-equol exposure has an on ER on ER expression in 3T3-L1 cells. information indicated that S-equol exposure has an impact effect expression in 3T3-L1 cells.Appl. Sci. 2021, 11, 9657 PEER Review Appl. Sci. 2021, 11, x FOR9 of 16 9 ofFigure 6. Effect of S-equol on the expression of estrogen receptors (A) and (B). 3T3-L1 fibroblasts Figure 6. Impact of S-equol on the expression of estrogen receptors (A) and (B). 3T3-L1 fibrotreated with S-equol (10) for 72 h wereh were induced to differentiation, and mRNA of ERof blasts treated with S-equol (10 M) for 72 induced to differentiation, and mRNA levels levels (A) and ERand had been(B) have been determined by real-time qRT-PCR on and 7. Untreated cells have been employed ER (A) (B) ER determined by real-time qRT-PCR on days three days 3 and 7. Untreated cells as handle. as control. Information werefrom three experiments in triplicate and expressed as mean SD. were employed Data have been obtained obtained from 3 experiments in triplicate and expressed as All values werevalues have been normalized with respect to -Actin, plus the expressionexpression was imply SD. All normalized with respect to -Actin, plus the alter in alter in was calculated calculated and with handle cells devoid of remedy on day 3. Statistical analysis was performed and compared compared with manage cells without the need of therapy on day three. Statistical analysis was performed by one-way ANOVA with Tukey’s numerous comparison post hoc the GraphPad by one-way ANOVA with Tukey’s various comparison post hoc test employing test employing the Prism GraphPad Prism 0.0001 vs. manage on day manage on day three; 0.0001; control on day 7; software. p software program. p 0.0001 vs. three; p 0.0001; pp 0.05 vs. p 0.05 vs. manage on day 7; #### p 0.0001 S-equol on day 3 vs. S-equol on day 7. #### p 0.0001 S-equol on day 3 vs. S-equol on day 7.4. Discussion The su.