Figure 1.
The expression pattern of p300 in wild-type HDPCs.
(A) Real-time qPCR was performed to measure p300 mRNA levels in wild-type primary HDPCs and in HDPCs serially passaged one to seven times. The mRNA level of each product was normalized to GAPDH mRNA levels. (B) The protein expression level of p300 was assessed by western blotting analysis (right panel) and densitometric evaluation (left panel; expressed as the ratio of p300 to GAPDH). (C) p300 mRNA levels were measured in HDPCs undergoing odontoblastic differentiation. (D) p300 protein levels were assessed in HDPCs undergoing odontoblastic differentiation by western blotting analysis (right panel) and densitometric evaluation (left panel; expressed as the ratio of p300 to GAPDH). All results are presented as the means ± SD of three independent experiments. Procedures were performed as described in the text (n = 3). * Statistically significant difference relative to the control, P<0.05.
Figure 2.
The stable overexpression of p300 and p300-ΔHAT in HDPCs.
(A) real-time qPCR was performed to measure the relative levels of p300 and p300-ΔHAT mRNA after the transduction with lentiviral vectors. The level of each product was normalized to GAPDH mRNA levels. (B) Protein expression levels of p300 and p300-ΔHAT were assessed by western blotting analysis (right panel) and densitometric evaluation (left panel; expressed as ratio to GAPDH). The expression of GAPDH served as a control. All results are presented as the means ± SD of three independent experiments. Procedures were performed as described in the text (n = 3). * Statistically significant differences relative to the control, P<0.05.
Figure 3.
The effect of p300 on the proliferation of HDPCs.
(A) Cell growth curves of HDPC/p300, HDPC/p300-ΔHAT and HDPC/V cells were constructed with the results of the CCK8 assay. The growth curves showed that p300 does not have a significant effect on the proliferation of HDPCs. (B) The BrdU assay revealed no significant differences in the amount of DNA synthesized by HDPC/p300, HDPC/p300-ΔHAT and HDPC/V cells. Newly synthesized DNA is stained red by BrdU and nuclei are stained blue by DAPI. Procedures were performed as described in the text (n = 3). All results are presented as the means ± SD of three independent experiments.
Figure 4.
p300 regulates the expression of OCT4, NANOG and SOX2, whereas HAT mutant p300 suppresses the expression of NANOG and SOX2.
(A) Real-time qPCR estimation of the relative endogenous mRNA levels of OCT4, NANOG and SOX2 in HDPC/p300 and HDPC/V cells. The mRNA levels of each product were normalized to the mRNA levels of GAPDH. (B) Western blotting analysis (right panel) and densitometric evaluation (left panel; expressed as the ratio of protein levels to GAPDH levels) measuring the levels of OCT4, NANOG and SOX2 proteins in HDPC/p300 and HDPC/V cells. The expression of GAPDH was used as an internal control. (C) Measurement of the relative endogenous mRNA levels of NANOG and SOX2 in HDPC/V, HDPC/p300 and HDPC/p300-ΔHAT cells using real-time qPCR. The mRNA levels of each product were normalized to GAPDH mRNA levels. (D) The results were further confirmed by western blotting (right panel) and densitometric evaluation (left panel; expressed as ratio to GAPDH). GAPDH was used as an internal control. All results are expressed as the means ± SD of three independent experiments. Procedures were performed as described in the text (n = 3). * Statistically significant difference relative to the control, P<0.05.
Figure 5.
p300 increases the activities of the NANOG and SOX2 promoters.
(A) HDPCs were transiently transfected with 500 ng of the NANOG or SOX2 promoter construct and 5 µg of the p300 or p300-ΔHAT expressing vector. Cells were harvested and the activities of the promoter were measured after 24 hours of transfection. (B) HDPCs were transiently transfected with 500 ng of the NANOG or SOX2 promoter construct and increasing amounts (0–5 µg) of the p300-expressing vector. After 24 hours of transfection, the cells were harvested and the activities of the promoters were measured. Procedures were performed as described in the text (n = 3). All results are presented as the means ± SD of three independent experiments. * Statistically significant difference relative to the control, P<0.05.
Figure 6.
The overexpression of p300 regulates the expression of odontoblastic marker genes in HDPCs in normal growth medium or odontoblastic induction medium.
(A) Real-time qPCR examination of the relative endogenous mRNA levels of DMP-1, DSPP, DSP, OPN and OCN in cells with overexpressed p300 and in negative control cells cultured under normal culture conditions. The mRNA levels of each product were normalized to GAPDH mRNA levels. (B) The overexpression of p300 increases the expression of odontoblastic marker genes in HDPCs induced to differentiate. Total RNA was extracted from the induced cells. The endogenous mRNA expression of DMP-1, DSPP, DSP, OPN and OCN was measured by real-time qPCR on days 7 and 14. GAPDH was used as an internal control. All results are presented as the means ± SD of three independent experiments. Procedures were performed as described in the text (n = 3). * Statistically significant difference relative to the control, P<0.05.
Figure 7.
The overexpression of p300 increases ALP activity and mineral formation in HDPCs.
(A) HDPC/V, HDPC/p300 and HDPC/p300-ΔHAT cells were cultured for 3 days in normal growth medium or odontoblastic induction medium, and the ALP activity in these cells was measured. GM, normal growth medium; IM, odontoblastic induction medium. (B) The effect of p300 on the formation of mineralized nodules in HDPCs cultured in odontoblastic induction medium, as analyzed by alizarin red S staining on day 21(×100). a: HDPC/V; b: HDPC/p300; c: HDPC/p300-ΔHAT. Scale bar, 100 µm. d: The histogram shows the quantification of mineralization by densitometry and reveals that remarkable decreases in mineralization occurred in HDPC/p300 and HDPC/p300-ΔHAT cells relative to control cells. All results are presented as the means ± SD of three independent experiments. Procedures were performed as described in the text (n = 3). * Statistically significant difference relative to the control, P<0.05.
Figure 8.
CHIP assay shows that p300 binds to the promoter region of OCN and DSPP in HDPCs.
(A, B) Cells were cross-linked with formaldehyde. Chromatin was immunoprecipitated with anti-p300 or anti-H3K9Ac antibodies. The chromatin was eluted, reverse cross-linked, and the eluted DNA was analyzed by PCR.