Journal: Experimental and Therapeutic Medicine

Article Title: IL-1β promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells via the BMP/Smad pathway within a certain concentration range

doi: 10.3892/etm.2020.9065

Figure Lengend Snippet: Effect of IL-1β on the BMP/Smad signaling pathway in mouse bone marrow mesenchymal stem cells. (A) Levels of p-Smad1/5, overall Smad1, Smad5 and Smad4 at 7 days were examined via Western blot analysis. (B) Quantitative assay of p-Smad1/5/GAPDH. * P<0.05, ** P<0.01, *** P<0.001. p-, phosphorylated; IL, interleukin; BMP, bone morphogenetic protein.

Article Snippet: After incubation in 5% skimmed milk for 1 h at 25˚C to block non-specific binding, the membranes were incubated with primary antibodies (1:1,000 dilution) against Smad1, Smad5, Smad4 and phosphorylated (p)-Smad1/5 (cat. no. 12656; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.) for 8 h at 4˚C.

Techniques: Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: IL-1β promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells via the BMP/Smad pathway within a certain concentration range

doi: 10.3892/etm.2020.9065

Figure Lengend Snippet: Effect of TGF-β/Smad inhibitor on the osteogenic differentiation of MBMMSCs. MBMMSCs were treated with osteogenic differentiation medium in the presence of 0.1 ng/ml IL-1β along with the inhibitor of TGF-β/Smad LDN193189. (A) Western blot analysis results for the levels of p-Smad1/5, and total Smad1, Smad5 and Smad4 at 7 days. (B) Quantitative results of p-Smad1/5/GAPDH. (C) Entire plate views of ALP staining at 7 days and alizarin red staining at 21 days. (D) Quantitative evaluation of ALP activity and alizarin red staining results. (E) mRNAs expression levels of RUNX2, ALP, COL1A1, BSP, BMP2, OCN and OPN at 7 days. Expression levels were normalized to GAPDH. *** P<0.001. p-, phosphorylated; IL, interleukin; ALP, alkaline phosphatase; BMP, bone morphogenetic protein; OCN, osteocalcin; OPN, osteopontin; BSP, bone sialoprotein; RUNX2, runt-related transcription factor 2; COL1A1, type I collagen; MBMMSCs, mouse bone marrow mesenchymal stem cells.

Article Snippet: After incubation in 5% skimmed milk for 1 h at 25˚C to block non-specific binding, the membranes were incubated with primary antibodies (1:1,000 dilution) against Smad1, Smad5, Smad4 and phosphorylated (p)-Smad1/5 (cat. no. 12656; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.) for 8 h at 4˚C.

Techniques: Western Blot, Staining, Activity Assay, Expressing

Journal: Oncology Reports

Article Title: Anti-proliferative effect of honokiol on SW620 cells through upregulating BMP7 expression via the TGF-β1/p53 signaling pathway

doi: 10.3892/or.2020.7745

Figure Lengend Snippet: Effects of HNK on TGF-β1 and p53 in SW620 cells. (A) The effect of HNK on the protein levels of Smad1/5/9 and p-Smad1/5/9 in SW620 cells was evaluated by western blot and quantitative analysis (GAPDH was used as a loading control). (B) Western blot and (C) quantitative analyses were used to assess the effect of HNK on TGF-β1 expression in SW620 cells. The effect of HNK on p53 and p-p53 levels was determined by using (D) western blot assays and (E) quantitative analyses; the ratio of p-p53/total p53 was determined by quantitative analyses. The control was treated with 6 µl DMSO (equivalent to HNK 30 µM). *P<0.05, **P<0.01 vs. control groups. HNK, honokiol; TGF, transforming growth factor; p-, phosphorylated.

Article Snippet: SW620 cells were treated with 5 µm LY364947 at 37°C for 24 or 48 h. The primary antibodies used were as follows: GAPDH (cat. no. 10494-1-AP), Bad (cat. no. 10435-1-AP), Bcl-2 (cat. no. 60178-1-Ig), proliferating cell nuclear antigen (PCNA; cat. no. 10205-2-AP), BMP7 (cat. no. 12221-1-AP) and TGF-β1 (cat. no. 21898-1-AP; all from Proteintech Technology, Inc.); Smad1/5/9 (cat. no. sc-6031-R), phosphorylated (p)-Smad1/5/9 (cat. no. sc-12353), Smad2/3 (cat. no. sc-8332) and p-Smad2/3 (cat. no. sc-11769; all from Santa Cruz Biotechnology, Inc.); p53 (cat. no. A11232) and p-p53 (cat. no. AP0083; both from Abclonal Technology, Inc.).

Techniques: Western Blot, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Thymosin beta 4 is associated with RUNX2 expression through the Smad and Akt signaling pathways in mouse dental epithelial cells

doi: 10.3892/ijmm.2015.2118

Figure Lengend Snippet: The decrease in runt-related transcription factor 2 (RUNX2) expression and calcification induced by inhibitors of the Smad (LDN193189) and PI3K-Akt (triciribine) signaling pathways in the mDE6 cells. (A and B) The expression levels of (A) p-Smad1/5 and (B) p-Akt were decreased in the mDE6 cells treated with LDN193189 and triciribine, respectively, compared with the controls. (C and D) The protein expression of Runx2 was inhibited by treatment of the mDE6 cells with (C) LDN193189 and (D) triciribine. (E–G) The mRNA expression levels of (E) amelogenin, X-linked (Amelx), (F) ameloblastin (Ambn) and (G) enamelin (Enam) were decreased by treatment with LDN193189. (H) The mDE6 cells were incubated with calcified induction medium (CIM) containing LDN193189 for 10 days. Calcification was suppressed by treatment with LDN193189 for 10 days. (I–K) The mRNA expression levels of (I) Amelx, (J) Ambn and (K) Enam were decreased by treatment with triciribine. (L) The cells were incubated with CIM containing triciribine for 10 days. Calcification was suppressed by treatment with triciribine for 10 days. The data are the means ± SD from triplicate samples. * P<0.05 and ** P<0.01 vs. control by a one-way ANOVA with the Tukey-Kramer comparison test. ND, not detectable.

Article Snippet: LDN193189 [an inhibitor of the phosphorylated (p-) Smad1/5/8 pathway] was obtained from AdooQ BioScience (Irvine, CA, USA).

Techniques: Expressing, Protein-Protein interactions, Incubation, Control, Comparison

Journal: Molecular Medicine Reports

Article Title: (R) -dehydroxyabscisic alcohol β -D-apiofuranosyl-(1ˮ→6’)- β -D-glucopyranoside enhances the osteoblastic differentiation of ST2 cells via the BMP/WNT pathways

doi: 10.3892/mmr.2018.9690

Figure Lengend Snippet: DAG treatment activates the BMP signaling pathway. (A) DAG treatment increased the gene expression of Bmp2 and Bmp4 , as well as the (B) protein expression of p-Smad1/5/8. (C) BMP antagonist Noggin pretreatment significantly attenuated the DAG-mediated increase in ALP activity and (D) mineralization, as well as the (E) protein expression of p-Smad1/5/8. Data are expressed as the mean ± standard deviation (n=3). # P<0.05 vs. DAG alone; *P<0.05 and **P<0.01 vs. control. Samples were measured in triplicate and experiments were repeated three times. DAG, ( R )-dehydroxyabscisic alcohol β-D-apiofuranosyl-(1ˮ→6’)-β-D-glucopyranoside; BMP, bone morphogenetic protein; ALP, alkaline phosphatase; p-, phosphorylated; Smad, mothers against decapentaplegic homolog; ARS, Alizarin Red S.

Article Snippet: Following blocking with 5% dried skimmed milk for 1 h at room temperature, membranes were washed three times with PBS/T (PBS containing 0.1%/Tween-20) and incubated with the following primary antibodies: Anti-mothers against decapentaplegic homolog (Smad)1/5/8 (cat. no. sc-6031-R; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-phosphorylated (p)-Smad1/5/8 (cat. no. AB3848-I; 1:500; EMD Millipore, Billerica, MA, USA), anti-β-catenin (cat. no. sc-7199; 1:500, Santa Cruz Biotechnology, Inc.), anti-Runx2 (cat. no. sc-10758; 1:500; Santa Cruz Biotechnology, Inc.) and anti-β-actin (cat. no. ab8227; 1:5,000, Abcam, Cambridge, UK) at 4°C overnight.

Techniques: Expressing, Activity Assay, Standard Deviation