Effects of Pro-Hyp on cell proliferation in Foxo1 knockdown cells
We examined the effects of Pro-Hyp on cell proliferation in Foxo1-knockdown cells. Pro-Hyp did not affect MC3T3-E1 cell proliferation and knockdown of Foxo1 did not change the proliferation of MC3T3-E1 cells. Furthermore, cell proliferation did not change with or without Pro-Hyp in Foxo1-knockdown cells (Fig. 1a).
Effects of Pro-Hyp on ALP activity in Foxo1-knockdown cells
Next, we examined the effect of Pro-Hyp on alkaline phosphatase (ALP) activity to investigate Pro-Hyp influence on osteoblast differentiation in Foxo1-knockdown cells. Pro-Hyp increased ALP activity in control siRNA-transfected cells. ALP activity in Foxo1-knockdown cells was slightly but not significantly lower than in cells transfected with control siRNA. The addition of Pro-Hyp in Foxo1-knockdown cells increased ALP activity (Fig. 1b).
Effects of Pro-Hyp on gene expression in Foxo1-knockdown cells
We examined whether the effects of Pro-Hyp on osteogenic gene expression were mediated via the expression of Foxo1. PCR results confirmed that knockdown was successful, as the expression of Foxo1 was clearly reduced.
The addition of Pro-Hyp did not affect Foxo1 mRNA expression level (Fig. 2a). Knockdown of Foxo1 induced expression of osterix mRNA, but not that of Runx2, Col1α1 or osteocalcin mRNA (Fig. 2b–e). In this experiment, knockdown of Foxo1 did not affect the expression of Runx2.
The addition of Pro-Hyp in control siRNA-transfected cells increased the expression level of Runx2, osterix and Col1α1 mRNA, but not that of osteocalcin mRNA. Furthermore, Pro-Hyp increased the expression level of Runx2 mRNA, but not that of osterix, Col1α1 or osteocalcin mRNA in Foxo1-knockdown cells. Foxg1 mRNA expression was significantly increased in Pro-Hyp-treated cells (Fig. 2f). Furthermore, Foxg1 mRNA expression was increased in Foxo1-knockdown cells. Foxg1 mRNA expression was not changed with or without Pro-Hyp in Foxo1-knockdown cells.
Effects of Pro-Hyp on protein expression in Foxo1-knockdown cells
To examine the effect of Pro-Hyp on the expression of osteogenic proteins in Foxo1-knockdown cells, we used western blotting to evaluate the expression of Runx2, osterix, Col1α1 and osteocalcin as osteogenic proteins. We also examined the influence of Pro-Hyp on the expression of Foxo1 and Foxg1.
Western blot results confirmed that knockdown was successful, as the expression of Foxo1 was clearly reduced. The addition of Pro-Hyp did not affect the Foxo1 protein expression level. Knockdown of Foxo1 induced the expression of osterix and Foxg1 protein but did not affect Runx2, Col1α1 or osteocalcin protein levels. The addition of Pro-Hyp in control siRNA-transfected cells increased the expression levels of Runx2, osterix, Col1α1 and Foxg1 protein, but those of not Foxo1 or osteocalcin. Furthermore, Pro-Hyp increased the expression levels of Runx2 and Foxg1 protein, but not those of osterix, Col1α1 or osteocalcin in Foxo1 knockdown cells (Fig. 3).
Effects of Pro-Hyp on MC3T3-E1 cell proliferation in Foxg1-knockdown cells
Pro-Hyp treatment and knockdown of Foxo1 both increased Foxg1 mRNA expression. Therefore, we examined whether Foxg1 knockdown could change the effects of Pro-Hyp on MC3T3-E1 cell proliferation and differentiation.
First, we assessed whether Foxg1 knockdown in MC3T3-E1 cells could change cell proliferation. Knockdown of Foxg1 did not change the proliferation of MC3T3-E1 cells. Furthermore, Pro-Hyp addition did not change cell proliferation in Foxg1-knockdown cells (Fig. 4a).
Effects of Pro-Hyp on ALP activity under lower levels of Foxg1 in MC3T3-E1 cells
We examined the effect of Pro-Hyp on ALP activity in Foxg1-knockdown cells. ALP activity was significantly inhibited in Foxg1-knockdown cells compared with control siRNA-transfected cells. ALP activity did not change, with or without Pro-Hyp, in Foxg1 knockdown cells (Fig. 4b).
Effects of Pro-Hyp on gene expression in Foxg1-knockdown cells
Foxg1 mRNA was significantly lower in Foxg1 knockdown cells than in control siRNA-transfected cells (Fig. 5a). Foxg1 mRNA expression did not further change, with or without Pro-Hyp treatment, in Foxg1-knockdown cells. Runx2 and Col1α1 mRNA expression did not change in Foxg1-knockdown cells compared with the levels in control siRNA-transfected cells (Fig. 5b and d). Osterix and osteocalcin mRNA expression had significantly decreased in si-Foxg1-transfected cells compared with the levels in control siRNA-transfected cells. Osterix and osteocalcin mRNA expression did not change in the presence or absence of Pro-Hyp in Foxg1-knockdown cells (Fig. 5c and e). Foxo1 mRNA expression was increased in Foxg1-knockdown cells and did not change with or without Pro-Hyp treatment (Fig. 5f).
Effects of Pro-Hyp on protein expression in Foxg1-knockdown cells
We examined the effect of Pro-Hyp on protein expression in Foxg1-knockdown cells. Western blot analysis confirmed successful knockdown, as the expression of Foxg1 clearly decreased. Knockdown of Foxg1 decreased the expression of Runx2 and osterix but did not affect Col1α1 and osteocalcin protein levels. The addition of Pro-Hyp in control siRNA-transfected cells increased the expression levels of Runx2, osterix and Foxg1 protein levels, but not those of Col1α1, osteocalcin and Foxo1. Furthermore, Pro-Hyp did not affect the expression levels of Foxg1, Runx2, osterix, Col1α1, osteocalcin or Foxo1 in Foxg1-knockdown cells (Fig. 6).