p16/p53 expression and telomerase activity in immortalized human dental pulp cells

Figures & data

Figure 1 Optical phase contrast microphotographs showing the morphology tDPSCs and nDPSCs under phase contrast microscopy (x400) during the growth phase, day 3 (A and C) and at confluence, day 7 (B and D). Also shown is some immunopositive staining of tDPSCs and nDPSCs.

Figure 2 Relative fluorescent readings used to assess telomerase activity in tDPSCs and nDPSCs after the introduction of exogenous HTERT. The results are expressed as the means ± SD (n = 4, *p < 0.001).

Figure 3 β-galactosidase staining in cultured human dental pulp stem cells. Cells were stained and photographed at 100x (A and C) or 200x (B and D) magnification. (A and B) Early passage nDPS C showing > 80% positivity and (C and D) Late passage tDPSCs showing < 10% positivity for β-galactosidase staining both depicted in the graph above. Positive staining disappeared after re-plating tDPSCs.

Figure 4 RT-PCR results for odontoblastic differentiation genes (ALP, cbfa-1, Col-1, DMP-1, DSPP, Osteopontin and Osteocalcin) and growth regulating factors p16, p21 and p53. Lane A, tDPSCs and Lane B, nDPSCs. GAPDH was run as an internal control and water as the negative control for both groups.

Figure 5 Analyses of cell proliferation using a DNA assay (A) and cell mineralization by way of an ALP assay (B) in tDPS C and nDPS C cell groups. The experiments were performed in triplicate. The values are means of the absorbance readings at 490 nm ± SD (n = 4, *p < 0.001).

Table 1 Primer sequences and cycling conditions for the genes analyzed

Primer	Sequence Product	Length (bp)
ALP	F: 5′-CCA CGT CTT CAC ATT TGG TG-3′ R: 5′-AGA CTG CGC CTG GTA GTT GT-3′	196 bp
β-ACTIN	F: 5′-ATG AGG ATG CTC ACG GAG CGC GGC TAC AGC-3′ R: 5′-ACA CCA CTG TGT TGG CGT ACA GGT CTT TGC-3′	331 bp
CBFA-1	F: 5′-TGA GAG CCG CTT CTC CAA CC-3′ R: 5′-GCG GAA GCA TTC TGG AAG GA-3′	270 bp
COL-1	F: 5′-CCA AAT CTG TCT CCC CAG A-3′ R: 5′-TCA AAA ACG AAG GGG AGA T-3′	213 bp
DSPP	F: 5′-TCA CAA GGG AGA AGG GAA TG-3′ R: 5′-TGC CAT TTG CTG TGA TGT TT-3′	182 bp
DMP-1	F: 5′-CAG GAG CAC AGG AAA AGG AG-3′ R: 5′-CTG GTG GTA TCT TGG GCA CT-3′	213 bp
GAPDH	F: 5′-ACC ACA GTC CAT GCC ATC AC-3′ R: 5′-TCC ACC ACC CTG TTG CTG TA-3′	452 bp
hTERT	F: 5′-CCT CTG TGC TGG GCC TGG ACG ATA-3′ R: 5′-ACG GCT GGA GGT CTG TCA AGG TAG-3′	282 bp
OCN	F: 5′-ATG AGA GCC CTC ACA CTC CT-3′ R: 5′-CAA GGG GAA GAG GAA AGA AG-3′	377bp
OPN	F: 5′-CAG TGA CCA GTT CAT CAG ATT CAT C-3′ R: 5′-CTA GGC ATC ACC TGT GCC ATA CC-3′	374bp
p16	F: 5′-GAC ATC CCC GAT TGA AAG AA-3′ R: 5′-TTT ACG GTA GTG GGG GAA GG-3′	198 bp
p21	F: 5′-GAC ACC ACT GGA GGG TGA CT-3′ R: 5′-CAG GTC CAC ATG GTC TTC CT-3′	172 bp
p53	F: 5′-GGC CCA CTT CAC CGT ACT AA-3′ R: 5′-GTG GTT TCA AGG CCA GAT GT-3′	156 bp