Effects of Tβ4 activation on BMP pathway in HDPCs.

<p>Cells were incubated <b>(A)</b> without or <b>(B)</b> with Tβ4 peptide in OM. <b>(A)</b> Expression of the BMP2 and BMP4 was examined by RT-PCR for 14 days. <b>(B)</b> Phosphorylation of Smad1/5/8 and Smad 2/3 was determined by Western blot analysis for 180 min. Tβ4 peptide increased expression of BMP2 and BMP4, phosphorylation of Smad1/5/8 and Smad2/3. Data are representative of three independent experiments.</p

Effects of ILK silencing on Tβ4 activation-induced odontogenic differentiation of HDPCs.

<p>Cells were transiently transfected with ILK siRNA (30 nM) for 24 h and then cultured in OM for 7 days <b>(A–C)</b> or incubated for 180 min in OM <b>(D, E)</b>. Differentiation was assessed by <b>(A)</b> measuring ALP activity (N = 6) and <b>(B)</b> Alizarin Red staining for 7 days. Expression of <b>(C)</b> BMP2, BMP4, Smad, <b>(D)</b> MAPK, and <b>(E)</b> transcription factors were determined by RT-PCR and Western blot analysis for 180 min. ILK siRNA blocked Tβ4-induced odontoblastic differentiation, and activation of the BMP, MAPK, and transcription factor pathways. ^*<i>P</i><0.05 compared to the control; ^#<i>P</i><0.05 compared with OM alone. Data are representative of three independent experiments.</p

Involvement of integrin and integrin-mediated signaling on effects of Tβ4 activation in HDPCs.

<p>Cells were incubated <b>(A)</b> without or <b>(B)</b> with Tβ4 peptide in OM. Expression of integrin (A) and its downstream signaling molecules (B) were determined by RT-PCR and Western blot analysis for 180 min and 14 days, respectively. The expression of integrin and its downstream signaling molecules including p-FAK, p-paxillin, and ILK were increased by Tβ4 peptide. Data are representative of three independent experiments.</p

Effects of Tβ4 activation on cell growth and odontogenic differentiation of HDPCs.

<p>Cells were treated with Tβ4 peptide in OM containing for 14 days. <b>(A)</b> Cell proliferation was determined by viable cell counting (N = 5). Differentiation was assessed by <b>(B)</b> measuring ALP activity (N = 6), <b>(C)</b> RT-PCR, and <b>(D)</b> Alizarin Red staining for 7 and 14 days. Tβ4 activation with a Tβ4 peptide promotes odontogenic differentiation, but not affects cell growth. ^*<i>P</i><0.05 compared to the control; ^#<i>P</i><0.05 compared with OM alone. Data are representative of three independent experiments.</p

Tβ4 mRNA and protein expression during odontoblastic differentiation of HDPCs.

<p>Cells were cultured in OM containing 10 mM β-glycerophosphate, 50 µg/ml ascorbic acid, and 100 nM dexamethasone for 0, 1, 3, 7 or 14 days. <b>(A)</b> Tβ4 mRNA expression was followed by RT-PCR relative to GAPDH expression (top) and by Western blotting relative to β-active expression (bottom). mRNA and protein expression of Tβ4 is upregulated during odontogenic differentiation in HDPCs. Effects of Tβ4 silencing on OM-induced growth and odontogenic differentiation of HDPCs <b>(B–F).</b> Cells were transiently transfected with Tβ4 siRNA (30 nM) for 24 h and then cultured in OM for 7 days. <b>(B)</b> Tβ4 knockdown by Tβ4 siRNA was detected by RT-PCR and Western blotting. <b>(C)</b> Cell proliferation was determined by viable cell counting (N = 5) for 7 days. Differentiation was assessed by <b>(D)</b> measuring ALP activity (N = 6), <b>(E)</b> RT-PCR, and <b>(F)</b> Alizarin Red staining for 7 days. Transfection with Tβ4 siRNA decreases OM-induced odontoblastic differentiation but not affected cell growth. ^*<i>P</i><0.05 compared to the control; ^#<i>P</i><0.05 compared with OM alone. Data are representative of three independent experiments.</p

Effects of a BMP inhibitor on Tβ4 activation-induced odontogenic differentiation.

<p><b>(A–C)</b> Cells were pretreated with noggin (1 µM) for 1 h, then incubated with Tβ4 (1 µg/ml) and BMP2 (100 ng/ml) for 14 days <b>(A–C)</b> or incubated for 180 min in OM <b>(D, E)</b>. Differentiation was assessed by <b>(A)</b> measuring ALP activity (N = 6) and <b>(B)</b> Alizarin Red staining for 14 days. Expression of <b>(C)</b> BMP2, BMP4, Smad, <b>(D)</b> MAPK, and <b>(E)</b> transcription factors were determined by RT-PCR and Western blot analysis for 180 min. Noggin blocked Tβ4 peptide-induced odontoblastic differentiation via Tβ4 peptide-induced phosphorylation of Smad1/5/8 and Smad2/3, and phosphorylation of MAPK and transcriptional factors. ^*<i>P</i><0.05 compared to the control; ^#<i>P</i><0.05 compared with OM alone. Data are representative of three independent experiments.</p

Effects of Tβ4 activation on the expression of MAPK pathways and transcription factors in HDPCs.

<p>Cells were incubated without or with Tβ4 peptide (1 µg/ml) in OM. <b>(A)</b> Phosphorylation of p38, JNK, and ERK MAPK was determined by Western blot analysis for 180 min. <b>(B)</b> Expression of the transcription factors Runx2 and Osterix was examined by RT-PCR for 180 min. Tβ4 activation enhanced the phosphorylation of p38, JNK, and ERK, and expression of transcription factors such as Runx2 and osterix. Data are representative of three independent experiments.</p

Reverse transcriptase-polymerase chain reaction (RT-PCR) primers and conditions.

<p>Reverse transcriptase-polymerase chain reaction (RT-PCR) primers and conditions.</p

Iron chelator induces IL-8 protein secretion (A, C) and IL-8 mRNA accumulation (B, D) in IHOK and HN12 cells in a time-dependent manner

<p><b>Copyright information:</b></p><p>Taken from "Differential regulation of iron chelator-induced IL-8 synthesis via MAP kinase and NF-κB in immortalized and malignant oral keratinocytes"</p><p>http://www.biomedcentral.com/1471-2407/7/176</p><p>BMC Cancer 2007;7():176-176.</p><p>Published online 13 Sep 2007</p><p>PMCID:PMC2078595.</p><p></p> Cells were incubated with DFO (1.0 mM) or IL1-β (10 ng/ml) for the indicated time periods. Levels of IL-8 protein and mRNA were determined by ELISA and semiquantitative RT-PCR, respectively. Numbers below the gels represent the intensity of IL-8 mRNA relative to GAPDH mRNA. These data are representative of three independent experiments

Iron chelator induced phosphorylated IκB-α in IHOK and HN12 cells on time dependent

<p><b>Copyright information:</b></p><p>Taken from "Differential regulation of iron chelator-induced IL-8 synthesis via MAP kinase and NF-κB in immortalized and malignant oral keratinocytes"</p><p>http://www.biomedcentral.com/1471-2407/7/176</p><p>BMC Cancer 2007;7():176-176.</p><p>Published online 13 Sep 2007</p><p>PMCID:PMC2078595.</p><p></p> Cells were treated with DFO (1.0 mM) or IL-1α (10 ng/ml) for the indicated time periods. Levels of IκB-α, p IκB-α were determined by Western blotting. The protein fraction was extracted, electrophoresed, transferred to membrane and blotted with respective antibodies. These data are representative of three independent experiments