Journal: Smart Materials in Medicine

Article Title: Eggshell-derived amorphous calcium phosphate: Synthesis, characterization and bio-functions as bone graft materials in novel 3D osteoblastic spheroids model

doi: 10.1016/j.smaim.2023.04.001

Figure Lengend Snippet: Fig. 10. Confocal microscopic inspection of OPN and collagen I expression of MC-3T3-E1 cells in 3D spheroids with ACP particles embedded and 2D culture system, respectively. (A) Left panel: OPN and collagen I expression in different culture media in 3D reconstructed images; Right panel: maximal fluorescent projection of OPN and collagen I in 3D spheroids as shown in left penal; (B) OPN and collagen I expression in different culture media in a 2D culture system. Scale bar ¼ 100 μm.

Article Snippet: In brief, cells and spheroids were cultured for 7 days and then fixed with 4 wt % PFA for 20 min. After washing in PBS thrice, samples were treated with 1% (v/v) TritionX-100 (X100, Sigma-Aldrich, USA) for 20 min, followed by blocking in 1 wt % BSA/PBS solution for 1 h. Then, the samples were incubated in goat-anti-mouse OPN primary antibody (Ab) (AF808, R&D systems, USA) and rabbit-anti-mouse collagen I Ab (ab21286, Abcam, USA) at 4 C overnight.

Techniques: Expressing

Journal: Journal of structural biology

Article Title: Nanostructure of mouse otoconia.

doi: 10.1016/j.jsb.2020.107489

Figure Lengend Snippet: Figure 6. Immunogold labeling of OPN in otoconia. (a, c) In wild-type mice, otoconial voids shown by TEM and SEM, respectively, after immunolabeling for OPN (aqueous procedures dissolve the calcitic otoconia). Inset: Intact wild-type mouse otoconia (without aqueous exposure) shown by SEM. By TEM (b) and SEM (d), immunogold labeling for OPN shows gold particles (arrows) at the surface of otoconial voids. (e, g) In OPN-deficient mice, otoconial voids shown by TEM and SEM, respectively, after immunolabeling for OPN. Inset: Intact otoconia from OPN-deficient mice (without aqueous exposure) shown by SEM. By TEM (f) and SEM (h), as expected in this negative control, immunogold labeling for OPN was absent.

Article Snippet: Otoconia-containing thin sections and block faces were incubated with anti-mouse OPN antibody (R&D Systems Oakville, Canada) for 1 h followed by washing and incubation with protein A-colloidal gold complex (14-nm gold particles, from G. Posthuma, University of Utrecht) for 1 h to detect the immunolabeling reactions.

Techniques: Labeling, Immunolabeling, Negative Control

Journal: International journal of molecular sciences

Article Title: Loss of Tyrosine Phosphatase Mu Promotes Scoliosis Progression Through Osteopontin-α5β1 Integrin Signaling and PIPK1γ90 Activity.

doi: 10.3390/ijms26031042

Figure Lengend Snippet: Figure 6. Loss of PTPµ contributes to increase the affinity of α5β1 integrin toward OPN. (A) Total RNA was extracted from osteoblasts of bipedal wild-type (WT) and PTPµ −/−mice, and mRNA expression levels of CD44 and indicated beta (β) alpha (α) integrins were examined by qPCR analysis using β-actin as the internal control. Error bars show standard error of the mean of three independent experiments performed in duplicate. (B) WT and PTPµ −/−osteoblasts (samples from 3 mice per genotype were pooled (n = 3)) were lysed and 20 µg of proteins were resolved by 10% SDS-PAGE and immunoblotted for antibodies specific for the indicated proteins. OPN served as the loading control. (C) WT and PTPµ −/−osteoblasts were lysed and subjected to immunoprecipitation (samples from 3 mice per genotype were pooled (n = 3)) with antibodies directed against each integrin subunit as indicated, including CD44 and OPN, and immunoprecipitates were resolved by 10% SDS-PAGE. Western blots were revealed with an antibody raised against OPN. OPN served as the loading control. The molecular weights for the integrins β1, β3, β5, β8, α1, α4, α5, αv, CD44, OPN are respectively 138kDa, 125kDa, 100kDa, 97kDa, 150kDa, 150kDa, 150kDa, 135kDa, 81kDa and 66kDa. (D) MC3T3- E1 cells (mouse osteoblasts) treated with PBS or rOPN were subjected to immunoprecipitation with antibodies against the Gi1, Gi2, or Gi3 alpha subunit. Precipitates were resolved by 10% SDS-PAGE and immunoblotted with an antibody directed against phospho-serine. Cells were pre-treated with antibody against β1 integrin for 30 min followed by an 18 h incubation with 0.5 µg/mL rOPN, prior to immunoprecipitation and immunoblotting. Bands shown are representative of results obtained with independent experiments. A second replicative experiment is shown in Supplementary Figure S6.

Article Snippet: The supernatant was then incubated with anti-PTPμ antibody (SC25433), (Santa Cruz Biotechnology Inc., Dallas, TX, USA) or anti- OPN antibody (sc10593; Santa Cruz Biotechnology, Inc.), followed by 1 h incubation with protein A beads with gentle rocking.

Techniques: Expressing, Control, SDS Page, Immunoprecipitation, Western Blot, Incubation

Journal: International journal of molecular sciences

Article Title: Effects of Red LED Irradiation in Enhancing the Mineralization of Human Dental Pulp Cells In Vitro.

doi: 10.3390/ijms24119767

Figure Lengend Snippet: Figure 3. Red LEDI in the mineralization stage enhanced the calcification of HDPCs. (A) Compared to the Nega group, mineralized deposition increased in the Posi and Mine groups but decreased in the Pro group. The Posi and Mine groups showed upregulation in the expression of DSPP, DMP1, OPN, and LAMP1. The Pro group showed increased expression of LAMP1 and decreased expression of DSPP, but DMP1 and OPN were unaffected. The Diff group showed enhanced LAMP1 expression but no change in DSPP, DMP1, and OPN expression. Compared to the Posi group, the Mine group showed similar mineralized nodule formation and odontogenic relative proteins expression (DSPP DMP1, LAMP1, and OPN), which were significantly reduced in the Pro and Diff groups. (B–F) Quantitative analysis of ARS staining and DSPP, DMP1, LAMP1, and OPN expression. (Results were normalized to the Nega group. * p < 0.05 compared to the Nega group, # p < 0.05 compared to the Posi group; DMP1: dentin matrix protein 1; LAMP1: lysosomal-associated membrane protein 1; OPN: osteopontin.)

Article Snippet: The antibody for osteopontin (OPN) was purchased from Novus.

Techniques: Expressing, Staining, Membrane