Journal: Kidney international

Article Title: Vitamin D receptor agonists increase klotho and osteopontin while decreasing aortic calcification in mice with chronic kidney disease fed a high phosphate diet

doi: 10.1038/ki.2012.322

Figure Lengend Snippet: (A) Arterial medial osteopontin (OPN) levels were increased by VDRA treatment. (i) OPN expression was low but detectable in the aortic media of CKD mice fed a high phosphate diet (CKD+HP). VDRA treatment increased smooth muscle cell expression of OPN in the aortic media in (ii) CKD+HP+C30 animal; and (iii) CKD+HP+P300 animal. Arrows point to aortic medial cells expressing OPN. Scale bars are 30 μm and objective is 40X. (B) Quantitation of OPN immunostaining showed no OPN expression in aortas from non-CKD controls, weak staining in CKD high phosphate-fed mice, and increased levels in VDRA-treated CKD mice (mean ± s.e.m., n =3 for all except CKD+HP+paricalcitol, n =5 where P100 and P300 samples were grouped). (C) Treatment of cultured VSMCs with 50 nM paricalcitol increased OPN levels in the media (significantly higher levels by ELISA at 48hr compared to time zero, ** P <0.001). Klotho (2 ng/mL) with/without FGF23 (2 ng/mL) did not upregulate OPN secretion. Three wells were sampled per time-point per treatment group, data are mean ± s.e.m. EtOH = ethanol control, KL = klotho, P50 = paricalcitol 50 nM.

Article Snippet: The following assays were used: QuantiChromTM Urea Assay Kit (BioAssay Systems, Hayward, CA) for BUN; o -cresolphthalein complexone kit from Teco Diagnostics (Anaheim, CA) for calcium; standard bioanalyzer at Phoenix Central Laboratory (Everett, WA) for phosphate; mouse FGF23 C-terminus ELISA kit (Immutopics, San Clemente, CA); DuoSet mouse osteopontin ELISA kit (R&D Systems, Minneapolis, MN); mouse intact PTH enzyme-linked immunosorbent assay kits (ALPCO Diagnostics, Salem, NH and Immutopics).

Techniques: Expressing, Quantitation Assay, Immunostaining, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Bioactive Materials

Article Title: Dual-function thermoresponsive antibiotic-loaded hydrogel with antimicrobial and osteogenic properties for implant-related infection control

doi: 10.1016/j.bioactmat.2026.02.044

Figure Lengend Snippet: In vivo immunohistochemical analysis (n = 6). (A) Representative immunolabeling images of the Control, PNVCL, and PNVCL/TC 25 mg/mL groups showing osteopontin (OPN) and osteocalcin (OCN) expression (immunopositive cells indicated by black arrows). (B) Mean scores (0–3) ± standard deviation for OPN immunostaining. (C) Mean scores (0–3) ± standard deviation for OCN immunostaining. Bars indicate statistically significant differences between groups (p < 0.05; Tukey HSD test).

Article Snippet: Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide for 1 h, followed by blocking of nonspecific binding sites with 1% bovine serum albumin for 12 h. The sections were then incubated with goat anti-osteopontin and goat anti-osteocalcin primary antibodies (sc-21742 and sc-30044, respectively; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: In Vivo, Immunohistochemical staining, Immunolabeling, Control, Expressing, Standard Deviation, Immunostaining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis.

doi: 10.4049/jimmunol.171.2.1085

Figure Lengend Snippet: FIGURE 2. Purification of murine OPN. A, OPN was purified from the serum-free conditioned medium of FGF2-T-MAE cells (see Materials and Methods). Mono-Q FPLC fractions (F) were tested (2.5 l/well) for the capacity to mediate the adhesion of MAE cells to nontissue culture plastic (E). B, Ten-microliter aliquots of fractions 14–17 were run on SDS-PAGE. Then total proteins were highlighted by Coomassie blue staining of the gel (a) or transferred to a nitrocellulose membrane and probed with anti-OPN Ab in a Western blot (b). C, Polystyrene nontissue culture microtiter plates were coated with purified OPN (fraction 15). Then, MAE cell adhesion was evaluated in the absence of any competitor (a), 10 g/ml of neutralizing anti-OPN Ab (b), 30 M of the v3/v5 integrin antagonist SCH 221153 (c), or of the inactive analog SCH 216687 (d). Data are the mean SD of three determinations (, p 0.01). D, Eukaryotic-produced rOPN (b; 500 ng) and purified OPN (c) were immunoprecipitated (IPPT) with anti-OPN Ab (-OPN). Immunoprecipitates (b and c) and nonimmunoprecipitated rOPN (a) were then probed in a Western blot (WB) with the same anti- OPN Ab and with anti-phosphoserine Ab (-P-ser).

Article Snippet: Eukaryotic-produced mouse recombinant OPN (rOPN) and neutralizing anti-mouse OPN Ab were obtained from R&D Systems (Minneapolis, MN).

Techniques: SDS Page, Staining, Membrane, Western Blot, Produced, Immunoprecipitation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis.

doi: 10.4049/jimmunol.171.2.1085

Figure Lengend Snippet: FIGURE 3. Recombinant FGF induces OPN up-regulation in MAE cells. MAE cells were incubated under serum-free conditions with 100 ng/ml of rFGF2. At different time points, RNA was extracted and RT-PCR was performed using specific murine OPN primers. Murine GAPDH prim- ers were used for the loading controls. Data are expressed as the OPN/ GAPDH ratio of the intensity of the corresponding PCR bands (A). In parallel, conditioned media collected at different time points from control () or rFGF2-treated () cell cultures were analyzed by Western blotting using affinity-purified anti-mouse OPN Abs (B). C, Cells were incubated under serum-free conditions for 48 h with the indicated stimuli (all at 100 ng/ml). At the end of incubation, conditioned media were collected and analyzed by Western blotting using affinity-purified polyclonal anti-mouse OPN Abs.

Article Snippet: Eukaryotic-produced mouse recombinant OPN (rOPN) and neutralizing anti-mouse OPN Ab were obtained from R&D Systems (Minneapolis, MN).

Techniques: Recombinant, Incubation, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis.

doi: 10.4049/jimmunol.171.2.1085

Figure Lengend Snippet: FIGURE 4. Recombinant FGF2 induces endothelial OPN up-regulation in vivo. A–D, Gelatin sponges adsorbed with vehicle (A), 1.0 g of rFGF2 (B and D), or 1.0 g of VEGF165 (C) were loaded onto the CAM of 8-day embryos. CAM fragments underneath the gelatin sponges were decorated with affinity-purified goat polyclonal anti-OPN Ab on day 12 or assessed by RT-PCR for OPN expression on day 9. OPN immunoreactivity is evi- dent in newly formed blood vessels (arrowheads) of rFGF2-treated CAMs (B) and limited to the basement membrane of the chorionic epithelium and stroma in control (A) and VEGF165-treated (C) CAMs. No specific signal was observed in rFGF2-treated CAMs in which the primary Ab was re- placed by preimmune rabbit serum (D). RT-PCR for OPN expression was performed using specific chicken OPN primers in CAMs treated with ve- hicle (), rFGF2 (F), or VEGF165 (V; inset in D). Chicken GAPDH prim- ers were used for the loading controls. E and F, Control and FGF2/Matrigel plugs were implanted s.c. into the flank of female C57BL/6 mice, After 7 days gels were collected and frozen, and 5-m sections were processed for OPN immunostaining and nuclear counterstaining. Note the strong OPN signal present in the endothelium lining the numerous blood vessels (arrows) of FGF2/Matrigel plugs (F) that were absent in control plugs (E). Infiltrating mononuclear cells were also evident in FGF2/Matrigel plugs (F).

Article Snippet: Eukaryotic-produced mouse recombinant OPN (rOPN) and neutralizing anti-mouse OPN Ab were obtained from R&D Systems (Minneapolis, MN).

Techniques: Recombinant, In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing, Membrane, Control, Immunostaining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis.

doi: 10.4049/jimmunol.171.2.1085

Figure Lengend Snippet: FIGURE 5. Recombinant OPN stimulates angiogenesis in the CAM. Vehicle (PBS), 1 g of rFGF2, or increasing concentrations of rOPN were delivered onto the CAM. After 4 days, blood vessels entering the implant were counted under a stereomicroscope. Data are the mean SD of 10 embryos (A). Implant and surrounding CAM specimens were dissected ex ovo and placed on a dark background. Images were then captured with a digital camera using a sidelight. Digitized 24-bit color images were then converted to 8-bit gray scale images, and their contrast was increased by 25% to highlight blood vessels using the Paint Shop Pro software (version 7.00; Jasco, Easton, MD). Note the highly vascularized appearance of rOPN implant (C) compared with the control sponge (B).

Article Snippet: Eukaryotic-produced mouse recombinant OPN (rOPN) and neutralizing anti-mouse OPN Ab were obtained from R&D Systems (Minneapolis, MN).

Techniques: Recombinant, Software, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis.

doi: 10.4049/jimmunol.171.2.1085

Figure Lengend Snippet: FIGURE 8. Angiogenic activity of RGD-OPN mutant. Gelatin sponges adsorbed with vehicle (PBS), rOPN, GST-OPN, or GST-RGD-OPN (500 ng/implant) were implanted onto the CAM (n 10). After 4 days, blood vessels entering the sponge were counted (A). Mononuclear cell infiltrate was assessed on histological sections of the CAMs by a planimetric method of point counting (B). Increasing concentrations of rOPN (F), GST-OPN (E), and GST-RGD-OPN (Œ) were evaluated for their chemotactic activity on freshly isolated human monocytes (C).

Article Snippet: Eukaryotic-produced mouse recombinant OPN (rOPN) and neutralizing anti-mouse OPN Ab were obtained from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Mutagenesis, Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis.

doi: 10.4049/jimmunol.171.2.1085

Figure Lengend Snippet: FIGURE 9. Angiogenic activity of OPN-treated human monocytes. Hu- man monocytes (m) were incubated for 48 h in the absence () or the presence (f) of 100 nM rOPN in serum-free RPMI. Then conditioned media were evaluated for their angiogenic activity in the CAM (3.0 l/ implant). Fresh medium with or without 100 nM rOPN was used as a negative control. , p 0.01 vs control. Inset, Monocytes were incubated for 4 h in the absence or the presence of 100 nM rOPN. Then RNA was extracted, and RT-PCR was performed using specific human TNF- and IL-8 primers. Human GAPDH primers were used for the loading controls.

Article Snippet: Eukaryotic-produced mouse recombinant OPN (rOPN) and neutralizing anti-mouse OPN Ab were obtained from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Incubation, Negative Control, Control, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Nanomedicine

Article Title: Effect of Attapulgite-Doped Electrospun Fibrous PLGA Scaffold on Pro-Osteogenesis and Barrier Function in the Application of Guided Bone Regeneration

doi: 10.2147/IJN.S244533

Figure Lengend Snippet: Sequence of Real-Time PCR

Article Snippet: For immunohistochemical staining, the decalcified sections were deparaffinized, hydrated with xylene and graded alcohols, and then incubated with primary Collagen-I (Col-I) (Source: goat, Southern Biotech, USA), OPN (Source: rabbit; 1:100 dilution; Novus Biologicals, USA), and OCN antibodies (Source: rat; 1:100 dilution; Abcam, USA).

Techniques: Sequencing

Journal: International Journal of Nanomedicine

Article Title: Effect of Attapulgite-Doped Electrospun Fibrous PLGA Scaffold on Pro-Osteogenesis and Barrier Function in the Application of Guided Bone Regeneration

doi: 10.2147/IJN.S244533

Figure Lengend Snippet: Real-time PCR analysis of Runx-2, ALP, OPN, and OCN mRNA relative expression (*p < 0.05). Abbreviations: PCR, polymerase chain reaction; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; OPN, osteopontin; OCN, osteocalcin.

Article Snippet: For immunohistochemical staining, the decalcified sections were deparaffinized, hydrated with xylene and graded alcohols, and then incubated with primary Collagen-I (Col-I) (Source: goat, Southern Biotech, USA), OPN (Source: rabbit; 1:100 dilution; Novus Biologicals, USA), and OCN antibodies (Source: rat; 1:100 dilution; Abcam, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction

Journal: International Journal of Nanomedicine

Article Title: Effect of Attapulgite-Doped Electrospun Fibrous PLGA Scaffold on Pro-Osteogenesis and Barrier Function in the Application of Guided Bone Regeneration

doi: 10.2147/IJN.S244533

Figure Lengend Snippet: Immunohistochemical analysis of new bone formation in each group 12 weeks post-implantation. Immunostaining of Col-I, OPN, and OCN demonstrated more intense brown staining in the bone and the surrounding fibroblastic-like tissue in the PLGA/ATT and Bio-Gide groups (scale bar = 100 μm). Abbreviations: Col-I, collagen-I; OPN, osteopontin; OCN, osteocalcin; PLGA, poly (lactic-co-glycolic acid) (PLGA); ATT, attapulgite.

Article Snippet: For immunohistochemical staining, the decalcified sections were deparaffinized, hydrated with xylene and graded alcohols, and then incubated with primary Collagen-I (Col-I) (Source: goat, Southern Biotech, USA), OPN (Source: rabbit; 1:100 dilution; Novus Biologicals, USA), and OCN antibodies (Source: rat; 1:100 dilution; Abcam, USA).

Techniques: Immunohistochemical staining, Immunostaining, Staining

Journal: Biomaterials

Article Title: 3D-printed oxygen-releasing scaffolds improve bone regeneration in mice.

doi: 10.1016/j.biomaterials.2021.121318

Figure Lengend Snippet: Fig. 6. O2 delivery from μtank scaffolds increases matrix and osteogenic protein deposition. Unloaded or O2-loaded 20% μtank scaffolds and ASCs were implanted into 4 mm murine calvarial defects for 8 weeks. (A, B) μCT of calvarial defects with scaffolds pseudocolored magenta and compared to empty defects in the contralateral side; scale bar = 4 mm. (C) Quantification of bone volume normalized to empty defect (n = 5), (D) Quantification of bone cross-sectional area compared to empty defect (n = 5), (E,F) H&E (inset boxes show where OPN immunostaining in I and J were taken from) and (G, H) Masson’s trichrome staining; Scale bar in E-H = 500 μm, (I, J) Immunofluorescence images of osteopontin staining; Scale bar = 50 μm, (K) Quantification of OPN immunostained area (n = 12), (L) Quantification of OPN signal intensity (n = 12). (*,*** represent p < 0.05, 0.001).

Article Snippet: Scaffolds were then incubated with primary antibodies for CD31 (R&D Systems, Minneapolis, MN, goat α mouse, AF3628), lamin A-C (Abcam, Cambridge, UK, rabbit α human, ab108595), osteopontin (R&D Systems, Minneapolis, MN, rat α human, MAB808-100), rabbit anti-CD86 (1:50; Thermo Fisher), goat anti-CD206 (1:50, Santa Cruz) in 10% normal goat serum in 0.2% Tween in PBS overnight, followed by three 5 min washes in PBS.

Techniques: Immunostaining, Staining, Immunofluorescence

Journal: Biomaterials

Article Title: 3D-printed oxygen-releasing scaffolds improve bone regeneration in mice.

doi: 10.1016/j.biomaterials.2021.121318

Figure Lengend Snippet: Fig. 8. O2 delivery from μtank scaffolds increases mineralization and osteogenic protein deposition in ectopic defects. Unloaded or O2-loaded 10% μtank scaffolds and ASCs were implanted into dorsal sub cutaneous defects for 8 weeks. (A) CT of calvarial defects; Scale bar = 1 mm. (B) Quantification of bone area compared to empty defect; dashed line is average initial scaffold bone volume (n = 8). (C) Immunoflu orescence images of osteopontin staining; scale bar = 100 μm. (D) Quantification of OPN immunostained area within the defects (n = 16–22). **, *** represent p < 0.01, 0.001).

Article Snippet: Scaffolds were then incubated with primary antibodies for CD31 (R&D Systems, Minneapolis, MN, goat α mouse, AF3628), lamin A-C (Abcam, Cambridge, UK, rabbit α human, ab108595), osteopontin (R&D Systems, Minneapolis, MN, rat α human, MAB808-100), rabbit anti-CD86 (1:50; Thermo Fisher), goat anti-CD206 (1:50, Santa Cruz) in 10% normal goat serum in 0.2% Tween in PBS overnight, followed by three 5 min washes in PBS.

Techniques: Staining

Journal: Aging (Albany NY)

Article Title: Increased expression of osteopontin in subchondral bone promotes bone turnover and remodeling, and accelerates the progression of OA in a mouse model

doi: 10.18632/aging.203707

Figure Lengend Snippet: sOPN was increased expressed in OA subchondral bone mainly in preosteoblasts and osteoblasts. ( A , B ) Western blot and qPCR analysis of the expression of OPN in primary osteoblasts of subchondral bone of an OA mouse model and control group; n ≥ 3. ( C , D ) Representative immunofluorescent and quantitative analysis of sOPN in OSX+ preosteoblasts or OCN+ osteoblasts in the tibial subchondral bone of an OA mouse model and control group. Positive cells were indicated with arrows. The boxed area is magnified in the corner, scale bars = 50 μm. Data are shown as mean ± s. d. and were analyzed by Student’s t test; n = 6, *P < 0.05,**P < 0.01 .

Article Snippet: We incubated sections with primary antibodies against Osterix (Santa Cruz Biotechnology, sc-393060), OPN (Proteintech, 22952-1-AP), β-tubulin (Proteintech, 10094-1-AP), OCN (Santa Cruz Biotechnology, sc-390877), CD31 (R&D Systems, AF3628), Endomucin (EMCN, Santa Cruz Biotechnology, sc-65495), MMP-13 (Affinity Biosciences Ltd., AF5355), collagen 10 (ABclonal, A6889), and p-AKT (Cell Signaling Technology, #4060) overnight at 4° C. For immunofluorescent staining, secondary antibodies conjugated with fluorescent tags were added and slides were incubated at room temperature for 1 h in the dark.

Techniques: Western Blot, Expressing, Control

Journal: Aging (Albany NY)

Article Title: Increased expression of osteopontin in subchondral bone promotes bone turnover and remodeling, and accelerates the progression of OA in a mouse model

doi: 10.18632/aging.203707

Figure Lengend Snippet: OPN regulates OA subchondral bone metabolism and accelerates subchondral bone remodeling. ( A ) Immunostaining of OCN+ cells in the tibial subchondral bone of an OA mouse model treated with vehicle, rmOPN or neutralizing antibody, and sham group. Positive cells were indicated with arrows, scale bars = 100 μm, n=6. ( B ) ALP staining and western blot analysis of collagen 1α (COL1α), RUNX2, and OCN expression of MC3T3-E1 cells treated with Dex (10 −7 M), β-glycerophosphate (10 mM) and VC (50 μg/ml) followed by the stimulation with rmOPN and OPN antibody for 3 or 5 days. ( C ) ALP activity of MC3T3-E1 cells treated with Dex (10 −7 M), β-glycerophosphate (10 mM) and VC (50 μg/ml) followed by the stimulation with rmOPN and antibody for 3 or 5 days, n=6. ( D ) Representative 3D reconstruction of micro-CT images of the tibial subchondral bone of an OA mouse model treated with vehicle, rmOPN or antibody, and sham group. Osteophyte were indicated with arrows, scale bar = 1 mm. Quantitative analysis of bone volume/total volume (BV/TV) and trabecular thickness (Tb. Th.), n=6. Data are shown as mean ± s. d. and were analyzed by one-way ANOVA, *P < 0.05, **P < 0.01 .

Article Snippet: We incubated sections with primary antibodies against Osterix (Santa Cruz Biotechnology, sc-393060), OPN (Proteintech, 22952-1-AP), β-tubulin (Proteintech, 10094-1-AP), OCN (Santa Cruz Biotechnology, sc-390877), CD31 (R&D Systems, AF3628), Endomucin (EMCN, Santa Cruz Biotechnology, sc-65495), MMP-13 (Affinity Biosciences Ltd., AF5355), collagen 10 (ABclonal, A6889), and p-AKT (Cell Signaling Technology, #4060) overnight at 4° C. For immunofluorescent staining, secondary antibodies conjugated with fluorescent tags were added and slides were incubated at room temperature for 1 h in the dark.

Techniques: Immunostaining, Staining, Western Blot, Expressing, Activity Assay, Micro-CT

Journal: Aging (Albany NY)

Article Title: Increased expression of osteopontin in subchondral bone promotes bone turnover and remodeling, and accelerates the progression of OA in a mouse model

doi: 10.18632/aging.203707

Figure Lengend Snippet: OPN promotes the formation of h-type vessels in subchondral bone of OA. ( A ) HUVECs were treated with rmOPN (100 ng/mL) and neutralizing antibody (1.0 μg/mL) for 24 h, and tubes were measured using a tube formation assay; scale bars = 100 μm, n = 6. ( B ) Representative images and quantitative analysis of Brdu (green) immunofluorescence in HUVECs treated with rmOPN (100 ng/mL) and neutralizing antibody (1.0 μg/mL) for 24 h; scale bars = 25 μm, n = 6. ( C , D ) Representative images and quantitative analysis of CD31 and Endomucin (EMCN) co-immunostaining in tibial subchondral bone of an OA mouse model treated with vehicle, rmOPN or neutralizing antibody, and sham group. Positive cells were indicated with arrows. Boxed area is magnified in the corner. Scale bars = 50 μm. Data are shown as mean ± s. d. and were analyzed by one-way ANOVA, n = 6, *P < 0.05, **P < 0.01 .

Article Snippet: We incubated sections with primary antibodies against Osterix (Santa Cruz Biotechnology, sc-393060), OPN (Proteintech, 22952-1-AP), β-tubulin (Proteintech, 10094-1-AP), OCN (Santa Cruz Biotechnology, sc-390877), CD31 (R&D Systems, AF3628), Endomucin (EMCN, Santa Cruz Biotechnology, sc-65495), MMP-13 (Affinity Biosciences Ltd., AF5355), collagen 10 (ABclonal, A6889), and p-AKT (Cell Signaling Technology, #4060) overnight at 4° C. For immunofluorescent staining, secondary antibodies conjugated with fluorescent tags were added and slides were incubated at room temperature for 1 h in the dark.

Techniques: Tube Formation Assay, Immunofluorescence, Immunostaining

Journal: Aging (Albany NY)

Article Title: Increased expression of osteopontin in subchondral bone promotes bone turnover and remodeling, and accelerates the progression of OA in a mouse model

doi: 10.18632/aging.203707

Figure Lengend Snippet: LY294002 inhibits OPN-mediated subchondral bone and cartilage degeneration in OA. ( C ) TRAP staining of osteoclasts and immunostaining of OCN+ cells were performed on subchondral bone of tibia in sham group, OA group and LY294002 treatment group. Positive cells were indicated with arrows, scale bars = 50 μm, n=6. Safranin O-fast green staining and OARSI scores of tibial articular cartilage in sham group, OA group and LY294002 treatment group. Scale bars = 200 μm. Representative 3D reconstruction of micro-CT images of tibial subchondral bone in sham group, OA group and LY294002 treatment group; scale bars = 1 mm. Quantitative analysis of bone volume/total volume (BV/TV) and trabecular thickness (Tb. Th.), n=6. Representative immunostaining and quantitative analysis of COL 10+, MMP-13+ cells in articular cartilage in sham group, OA group and LY294002 treatment group. Scale bars = 50 μm, n=6. Data are shown as mean ± s. d. and were analyzed by one-way ANOVA, *P < 0.05, **P < 0.01 .

Article Snippet: We incubated sections with primary antibodies against Osterix (Santa Cruz Biotechnology, sc-393060), OPN (Proteintech, 22952-1-AP), β-tubulin (Proteintech, 10094-1-AP), OCN (Santa Cruz Biotechnology, sc-390877), CD31 (R&D Systems, AF3628), Endomucin (EMCN, Santa Cruz Biotechnology, sc-65495), MMP-13 (Affinity Biosciences Ltd., AF5355), collagen 10 (ABclonal, A6889), and p-AKT (Cell Signaling Technology, #4060) overnight at 4° C. For immunofluorescent staining, secondary antibodies conjugated with fluorescent tags were added and slides were incubated at room temperature for 1 h in the dark.

Techniques: Staining, Immunostaining, Micro-CT

Journal: Critical Care

Article Title: Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury

doi: 10.1186/s13054-015-0782-3

Figure Lengend Snippet: Expression of OPN in lungs, spleen and plasma after sepsis in mice . Lungs, spleen and blood samples were harvested at 20 h after CLP or sham operation. Lung expression of OPN at its (A) RNA and (B) protein levels was measured by using real-time PCR and Western blot, respectively. OPN expression in the spleen tissues was determined at its (C) RNA and (D) protein levels. OPN expression in each sample was normalized to β-actin expression and the value of sham group was designated as one for comparison. (E) OPN expression in 3.0 μL of plasma from sham and CLP animals was determined by Western blot. Each blot was quantified by densitometry analysis. Representative blots against OPN and β-actin are shown. Data are expressed as means ± SEM (n = 5 mice/group) and compared by Student’s t test ( * P <0.05 vs. shams). CLP, cecal ligation and puncture; OPN, osteopontin; PCR, polymerase chain reaction; SEM, standard error of the mean.

Article Snippet: Recombinant mouse osteopontin (rmOPN) purchased from R&D Systems (Catalog No.: 441-OP) was used as a chemoattractant for dHL-60 cell migration. rmOPN was prepared by cloning the cDNA encoding Leu17 to Asn294 (Glu99Gly) of OPN transcript without its signal peptide sequence (Accession No.: Q547B5) into a C-terminal 6-His tag expression vector.

Techniques: Expressing, Clinical Proteomics, Real-time Polymerase Chain Reaction, Western Blot, Comparison, Ligation, Polymerase Chain Reaction

Journal: Critical Care

Article Title: Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury

doi: 10.1186/s13054-015-0782-3

Figure Lengend Snippet: Effect of anti-OPN Ab treatment on plasma levels of organ injury markers and proinflammatory cytokines and chemokine in CLP animals . Sepsis was induced in mice by CLP and anti-OPN Ab or non-immunized IgG control at a dose of 50 μg/mice in 100 μl volumes was injected through the jugular vein. In the vehicle group, 100 μl of PBS was injected in CLP mice via the jugular vein. Blood samples were drawn by cardiac puncture at 20 h of sham-operated, vehicle and anti-OPN Ab-treated mice for measuring (A) ALT, (B) AST and (C) LDH. Similarly, the blood samples collected at 20 h after CLP were measured for (D) IL-6, (E) IL-1β and (F) MIP-2 by ELISA. Data are expressed as means ± SEM (n = 5 mice/group) and compared by one-way ANOVA and SNK method ( * P <0.05 vs. shams; # P <0.05 vs. vehicle). Ab, antibody; ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate aminotransferase; CLP, cecal ligation and puncture; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; IL, interleukin; LDH, lactate dehydrogenase; MIP-2, macrophage inflammatory protein 2; OPN, osteopontin; PBS, phosphate-buffered saline; SEM, standard error of the mean; SNK, Student-Newman-Keuls.

Article Snippet: Recombinant mouse osteopontin (rmOPN) purchased from R&D Systems (Catalog No.: 441-OP) was used as a chemoattractant for dHL-60 cell migration. rmOPN was prepared by cloning the cDNA encoding Leu17 to Asn294 (Glu99Gly) of OPN transcript without its signal peptide sequence (Accession No.: Q547B5) into a C-terminal 6-His tag expression vector.

Techniques: Clinical Proteomics, Control, Injection, Enzyme-linked Immunosorbent Assay, Ligation, Saline

Journal: Critical Care

Article Title: Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury

doi: 10.1186/s13054-015-0782-3

Figure Lengend Snippet: Effect of anti-OPN Ab on the expression of proinflammatory cytokines and chemokine in the lungs after CLP . Mice were subjected to CLP and 100 μl of anti-OPN Ab (50 μg/mouse) or PBS as vehicle was injected through the jugular vein. Lung tissues were collected after 20 h from sham-operated, vehicle, and anti-OPN Ab-treated mice. The tissue expression of (A) IL-6, (B) IL-1β and (C) MIP-2 was determined by real-time PCR. Gene expression was normalized to β-actin. The sham expression level was designated as one for comparison. Similarly, 50 μg of total protein extracted from the lung tissues were examined for (D) IL-6, (E) IL-1β and (F) MIP-2 analysis by ELISA. Finally, the results are expressed as per mg of proteins. Data are represented as means ± SEM (n = 5 mice/group) and compared by one-way ANOVA and SNK method ( * P <0.05 vs. sham; # P <0.05 vs. vehicle). Ab, antibody; ANOVA, analysis of variance; CLP, cecal ligation and puncture; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; MIP-2, macrophage inflammatory protein 2; OPN, osteopontin; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SEM, standard error of the mean; SNK, Student-Newman-Keuls.

Article Snippet: Recombinant mouse osteopontin (rmOPN) purchased from R&D Systems (Catalog No.: 441-OP) was used as a chemoattractant for dHL-60 cell migration. rmOPN was prepared by cloning the cDNA encoding Leu17 to Asn294 (Glu99Gly) of OPN transcript without its signal peptide sequence (Accession No.: Q547B5) into a C-terminal 6-His tag expression vector.

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Gene Expression, Comparison, Enzyme-linked Immunosorbent Assay, Ligation, Saline, Polymerase Chain Reaction

Journal: Critical Care

Article Title: Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury

doi: 10.1186/s13054-015-0782-3

Figure Lengend Snippet: Evaluation of lung histology in mice after CLP . Lung tissues were harvested after 20 h from sham-operated, vehicle and anti-OPN Ab-treated mice and stained with H&E. Slides were observed under light microscopy at × 200 original magnifications (inset: ×400 original magnification). (A) Representative images for sham, vehicle, and anti-OPN Ab treatment groups are shown. (B) Histological injury scores of the lungs in different groups were quantified as described in the . Data are expressed as means ± SEM (n = 5 mice/group) and compared by one-way ANOVA and SNK method ( * P <0.05 vs. shams; # P <0.05 vs. vehicles). Ab, antibody; ANOVA, analysis of variance; CLP, cecal ligation and puncture; H&E, hematoxylin and eosin; OPN, osteopontin; SEM, standard error of the mean; SNK, Student-Newman-Keuls.

Article Snippet: Recombinant mouse osteopontin (rmOPN) purchased from R&D Systems (Catalog No.: 441-OP) was used as a chemoattractant for dHL-60 cell migration. rmOPN was prepared by cloning the cDNA encoding Leu17 to Asn294 (Glu99Gly) of OPN transcript without its signal peptide sequence (Accession No.: Q547B5) into a C-terminal 6-His tag expression vector.

Techniques: Staining, Light Microscopy, Ligation

Journal: Critical Care

Article Title: Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury

doi: 10.1186/s13054-015-0782-3

Figure Lengend Snippet: Assessment of neutrophil infiltration into the lungs after CLP . Lung tissues were collected at 20 h after sham-operation, vehicle and anti-OPN Ab treatment in mice. (A) Representative images of the lung tissue sections with immunostaining against Gr-1 at × 200 original magnification are shown. Arrows demarcate examples of areas of staining Gr-1-positive cells. (B) A graphical representation of Gr-1-positive cells averaged over 10 microscopic fields per animal lung tissues. (C) Myeloperoxidase (MPO) activities in lung tissues were determined spectrophotometrically. Data are expressed as means ± SEM (n = 5 mice/group) and compared by one-way ANOVA and SNK method ( * P <0.05 vs. shams; # P <0.05 vs. vehicles). Ab, antibody; ANOVA, analysis of variance; CLP, cecal ligation and puncture; OPN, osteopontin; SEM, standard error of the mean; SNK, Student-Newman-Keuls.

Article Snippet: Recombinant mouse osteopontin (rmOPN) purchased from R&D Systems (Catalog No.: 441-OP) was used as a chemoattractant for dHL-60 cell migration. rmOPN was prepared by cloning the cDNA encoding Leu17 to Asn294 (Glu99Gly) of OPN transcript without its signal peptide sequence (Accession No.: Q547B5) into a C-terminal 6-His tag expression vector.

Techniques: Immunostaining, Staining, Ligation

Journal: Critical Care

Article Title: Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury

doi: 10.1186/s13054-015-0782-3

Figure Lengend Snippet: rmOPN-mediated neutrophil migration in vivo and in vitro. C57BL/6 mice were injected with rmOPN at a dose of 2.5 μg/mice, intratracheally. After 20 h, cells from the lung tissues were isolated and stained with APC-anti-Gr-1 Ab and then subjected to flow cytometry. (A) Representative dot blots indicating the percentages of Gr-1-positive cells are shown. (B) The mean percentages of Gr-1-positive cells obtained from PBS- and rmOPN-injected mice are shown. Data are expressed as means ± SEM (n = 4 mice/group) and compared by Student’s t test ( * P <0.05 vs. PBS). (C) A total of 5 × 10 5 primary neutrophil cells isolated from mouse bone marrow were placed into the insert of a Boyden chamber. The bottom compartment contained the RPMI medium with PBS or rmOPN at a dose of 10 μg/ml as a chemotactic stimulus. After 2 h, the migrated primary neutrophil cells were counted. A representative image of the migrated primary neutrophil cells labeled with PI (red fluorescence) on the bottom of the transwell membrane is shown. Cells were observed at × 200 original magnification. (B) Migrated primary neutrophils were counted in five random microscopic fields per well and averaged in each group. Data are expressed as means ± SEM (n = 4/group) and compared by Student’s t test ( * P <0.05 vs. PBS). Ab, antibody; OPN, osteopontin; PBS, phosphate-buffered saline; PI, propidium iodide; rmOPN, recombinant mouse OPN; SEM, standard error of the mean.

Article Snippet: Recombinant mouse osteopontin (rmOPN) purchased from R&D Systems (Catalog No.: 441-OP) was used as a chemoattractant for dHL-60 cell migration. rmOPN was prepared by cloning the cDNA encoding Leu17 to Asn294 (Glu99Gly) of OPN transcript without its signal peptide sequence (Accession No.: Q547B5) into a C-terminal 6-His tag expression vector.

Techniques: Migration, In Vivo, In Vitro, Injection, Isolation, Staining, Flow Cytometry, Labeling, Fluorescence, Membrane, Saline, Recombinant

Journal: Critical Care

Article Title: Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury

doi: 10.1186/s13054-015-0782-3

Figure Lengend Snippet: rmOPN-mediated activation of FAK and MAP kinase signaling molecules in dHL-60 cells . Differentiated HL-60 cells were incubated with PBS or rmOPN at a dose of 2 μg/mL for 90 min. (A) The status of phosphorylated FAK (pFAK) and β-actin, (B) pERK and total ERK and (C) pp38 and total p38 in each group was determined by Western blot. Blots were scanned and quantified with densitometry. Representative blots against these proteins are shown. Data are expressed as means ± SEM obtained from two independent experiments (n = 5/group) and compared by Students t test (* P <0.05 vs. PBS). ERK, extracellular signal-regulated protein kinase; FAK, focal adhesion kinase; MAP, mitogen-activated protein; OPN, osteopontin; PBS, phosphate-buffered saline; rmOPN, recombinant mouse OPN; SEM, standard error of the mean.

Article Snippet: Recombinant mouse osteopontin (rmOPN) purchased from R&D Systems (Catalog No.: 441-OP) was used as a chemoattractant for dHL-60 cell migration. rmOPN was prepared by cloning the cDNA encoding Leu17 to Asn294 (Glu99Gly) of OPN transcript without its signal peptide sequence (Accession No.: Q547B5) into a C-terminal 6-His tag expression vector.

Techniques: Activation Assay, Incubation, Western Blot, Saline, Recombinant

Journal: Critical Care

Article Title: Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury

doi: 10.1186/s13054-015-0782-3

Figure Lengend Snippet: Effect of rmOPN on dHL-60 cell migration in vitro. Boyden chamber assay was performed as described in . (A) A representative image of migrated dHL-60 cells labeled with PI (red fluorescence) on the bottom of the transwell membrane. Cells were observed at × 200 original magnification. (B) Migrated dHL-60 cells were counted in five random microscopic fields per well and averaged in each group. Data are expressed as means ± SEM (n = 4/group) and compared by one-way ANOVA and SNK method ( * P <0.05 vs. PBS; # P <0.05 vs. rmOPN). ANOVA, analysis of variance; OPN, osteopontin; PBS, phosphate-buffered saline; PI, propidium iodide; rmOPN, recombinant mouse OPN; SEM, standard error of the mean; SNK, Student-Newman-Keuls.

Article Snippet: Recombinant mouse osteopontin (rmOPN) purchased from R&D Systems (Catalog No.: 441-OP) was used as a chemoattractant for dHL-60 cell migration. rmOPN was prepared by cloning the cDNA encoding Leu17 to Asn294 (Glu99Gly) of OPN transcript without its signal peptide sequence (Accession No.: Q547B5) into a C-terminal 6-His tag expression vector.

Techniques: Migration, In Vitro, Boyden Chamber Assay, Labeling, Fluorescence, Membrane, Saline, Recombinant

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Interferon-γ plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice.

doi: 10.1002/jbmr.350

Figure Lengend Snippet: Fig. 2. Markers of osteoblast differentiation in IFNgR1/ mice compared with IFNgR1þ/þ mice. (A, B) Immunostaining for Runx2 (A) and OPN (B) (brown staining) in 8-week-old IFNgR1/ and IFNgR1þ/þ mice. Note the reduction in both Runx2 and OPN expression in IFNgR1/ mice compared with IFNgR1þ/þ

Article Snippet: After blocking with PBS containing 0.1% Tween 20 and 10% nonfat dry milk, membranes were incubated overnight at 48C using mouse monoclonal antibodies directed against Runx2, OPN, and RANKL (1:1000; Santa Cruz Biotechnology, Inc.).

Techniques: Immunostaining, Staining, Expressing

Journal: Acta Histochemica et Cytochemica

Article Title: Expression and Dynamics of Podoplanin in Cultured Osteoblasts with Mechanostress and Mineralization Stimulus

doi: 10.1267/ahc.17031

Figure Lengend Snippet: Immunostaining of cultured osteoblasts subjected to elongation straining. The osteoblasts cultured in the mineralization medium were immunostained (red) by anti-podoplanin, anti-osteopontin, and andi-osteocalcin, and all staining intensities increased with the duration of the elongation straining (days). Nuclei were stained by DAPI. Bars = 100 μm.

Article Snippet: Contaminating genomic DNA was removed using DNAfree (Ambion, Huntingdon, UK), and the RT was performed on 30 ng of total RNA at 30°C for 10 min, 55°C for 30 min, and 99°C for 5 min, followed by 30 cycles of PCR for amplification (extension at 72°C for 30 sec, denaturation at 94°C for 30 sec, annealing at 60°C for 90 sec, and followed by final extension at 72°C for 10 min) using the Ex Taq hot start version (Takara Bio Inc., Otsu, Japan) with 50 pM of primer sets for mouse mRNA of β-actin (Mm02619580_g1; Thermo Fisher Scientific , Inc., Yokohama, JAPAN), podoplanin (Mm01348912_g1; Thermo Fisher Scientific), osteopontin (Mm00436767_m1; Thermo Fisher Scientific), and osteocalcin (Mm03413826_mH; Thermo Fisher Scientific), where the specificities had been confirmed by the manufacturer (Thermo Fisher Scientific).

Techniques: Immunostaining, Cell Culture, Staining

Journal: Acta Histochemica et Cytochemica

Article Title: Expression and Dynamics of Podoplanin in Cultured Osteoblasts with Mechanostress and Mineralization Stimulus

doi: 10.1267/ahc.17031

Figure Lengend Snippet: A. Ratio of the immunostained area in cultured osteoblasts subjected to elongation straining. The immunostained area of osteoblasts cultured in the mineralization medium was measured at five different locations in the images using Image J. The relative expressed amounts of each protein were estimated by the ratio of the immunostained area (%): podoplanin, osteopontin, and osteocalcin-positive area/area scanned in the culture. All of the relative expressed amounts of podoplanin, osteopontin, and osteocalcin increased with duration (days) of the elongation straining, and the amounts of culture for 2 and 3 days were statistically significantlly larger than in the unstrained culture. The relative amounts for podoplanin and osteocalcin were significantly larger than the culture at 1 day without straining. *Significantly different in ANOVA (P < 0.01). B. Real time-PCR analysis for podoplanin, osteopontin, and osteocalcin mRNAs in cultured osteoblasts exposed to elongation straining. The relative amounts of mRNAs were expressed by means of the ratio (%): podoplanin, osteopontin, and osteocalcin cDNA units/β-actin cDNA units. All of the relative amounts of mRNAs increased with duration (days) of elongation straining and reached a plateau within three days. The mRNA of podoplanin of osteoblasts cultured with straining in mineralization medium (closed circles) is significantly larger than in cells cultured with straining in non-mineralization medium (open squares) and without strain in mineralization medium (open triangles). The mRNA of podoplanin of osteoblasts cultured with straining in non-mineralization medium is significantly larger than cells cultured without straining in mineralization medium. The mRNA of osteopontin of osteoblasts cultured with straining for 2–5 days in mineralization medium (closed circles) is significantly larger than in cells cultured with straining in non-mineralization medium (open squares) and without straining in mineralization medium (open triangles). The mRNA of osteopontin of osteoblasts cultured with straining in non-mineralization medium is not significantly different from cells cultured without straining in mineralization medium. The mRNA of osteocalcin of osteoblasts cultured with straining for 3–5 days in mineralization medium (closed circles) is significantly larger than in cells cultured with straining in non-mineralization medium (open squares) and without straining in mineralization medium (open triangles). The mRNA of osteocalcin of osteoblasts cultured without straining in mineralization medium is significantly larger than in cells cultured with straining in non-mineralization medium. In the mineralization medium, the significant increase of osteocalcin mRNA occurred earlier in osteoblasts with straining than in the cells without straining. Values are mean ± SD of data from 6 wells for each group. *Significantly different in ANOVA (P < 0.01).

Article Snippet: Contaminating genomic DNA was removed using DNAfree (Ambion, Huntingdon, UK), and the RT was performed on 30 ng of total RNA at 30°C for 10 min, 55°C for 30 min, and 99°C for 5 min, followed by 30 cycles of PCR for amplification (extension at 72°C for 30 sec, denaturation at 94°C for 30 sec, annealing at 60°C for 90 sec, and followed by final extension at 72°C for 10 min) using the Ex Taq hot start version (Takara Bio Inc., Otsu, Japan) with 50 pM of primer sets for mouse mRNA of β-actin (Mm02619580_g1; Thermo Fisher Scientific , Inc., Yokohama, JAPAN), podoplanin (Mm01348912_g1; Thermo Fisher Scientific), osteopontin (Mm00436767_m1; Thermo Fisher Scientific), and osteocalcin (Mm03413826_mH; Thermo Fisher Scientific), where the specificities had been confirmed by the manufacturer (Thermo Fisher Scientific).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction

Journal: Acta Histochemica et Cytochemica

Article Title: Expression and Dynamics of Podoplanin in Cultured Osteoblasts with Mechanostress and Mineralization Stimulus

doi: 10.1267/ahc.17031

Figure Lengend Snippet: A. Antibody-dependent cytotoxicity test. The viability of osteoblasts cultured with rat anti-mouse podoplanin, rabbit anti-mouse osteopontin, rabbit anti-mouse osteocalcin, mouse anti-actin, and rat isotype control IgG were tested. Cells were cultured in mineralization medium containing 1 μg/ml (open bars) and 2 μg/ml (closed bars) of antibodies for 10 days, and cultured in mineralization medium containing 1 μg/ml of antibodies for 20 days (gray bars). The number of alive cells was estimated by optical density measurements at the 450 nm wavelength for the amount of formazan dye from reduced WST-8. The cell viability with antibodies were expressed by the absorbance ratio (%): absorbance of culture with antibodies/absorbance of culture without antibodies. There were no significant differences among numbers of alive osteoblasts cultured in mineralization medium without antibodies or with different antibodies at the same concentration and at the same culture duration in ANOVA (P < 0.01). Values are mean ± SD of data from 6 wells for each group. B. Mineralization assay. Alizarin red staining was performed on culture products from osteoblasts cultured in the mineralization medium containing 1 μg/ml of rat anti-mouse podoplanin, rabbit anti-mouse osteopontin, rabbit anti-mouse osteocalcin, mouse anti-actin, and rat isotype control IgG for 20 days. Alizarin red-reacted mineralization products were seen in the culture with no antibodies, and in cultures with mouse anti-actin and rat IgG, but there were no reaction products in cultures with antibodies for podoplanin, osteopontin, and osteocalcin. C. Amounts of mineralization products from osteoblasts cultured for 10 days. Alizarin red-reacted mineralization products from osteoblasts cultured with antibodies of 1 μg/ml (closed bars) and 2 μg/ml (open bars) concentrations were dissolved in formic acid and the mineralization was estimated by the optical density measurements at the 450 nm wavelength. Mineralization products were significantly less in cultures with antibodies for podoplanin, osteopontin, and osteocalcin than in culture with no antibodies and in cultures with mouse anti-actin, and rat isotype control IgG. There were no significant differences in cultures with no antibodies and with mouse anti-actin and rat isotype control IgG; in cultures with antibodies for podoplanin, osteopontin, and osteocalcin; in two cultures with the same antibody at two different concentrations. Values are mean ± SD of data from 6 wells for each group. *Significantly different from medium, anti-actin, and isotype controls in ANOVA (P < 0.01). D. Amounts of mineralization products from osteoblasts cultured for 20 days. Alizarin red-reacted mineralization products from osteoblasts cultured with antibodies of 1 μg/ml concentration were dissolved in formic acid and the mineralization was estimated by optical density measurements at the 450 nm wavelength. Mineralization products were significantly less in cultures with antibodies for podoplanin, osteopontin, and osteocalcin than cultures with no antibodies and cultures with mouse anti-actin, and rat isotype control IgG. There were no significant differences among cultures with no antibodies and with mouse anti-actin, and rat isotype control IgG; in cultures with antibodies for podoplanin, osteopontin, and osteocalcin. Values are mean ± SD of data from 6 wells for each group. *Significantly different from medium, anti-actin, and isotype control in ANOVA (P < 0.01).

Article Snippet: Contaminating genomic DNA was removed using DNAfree (Ambion, Huntingdon, UK), and the RT was performed on 30 ng of total RNA at 30°C for 10 min, 55°C for 30 min, and 99°C for 5 min, followed by 30 cycles of PCR for amplification (extension at 72°C for 30 sec, denaturation at 94°C for 30 sec, annealing at 60°C for 90 sec, and followed by final extension at 72°C for 10 min) using the Ex Taq hot start version (Takara Bio Inc., Otsu, Japan) with 50 pM of primer sets for mouse mRNA of β-actin (Mm02619580_g1; Thermo Fisher Scientific , Inc., Yokohama, JAPAN), podoplanin (Mm01348912_g1; Thermo Fisher Scientific), osteopontin (Mm00436767_m1; Thermo Fisher Scientific), and osteocalcin (Mm03413826_mH; Thermo Fisher Scientific), where the specificities had been confirmed by the manufacturer (Thermo Fisher Scientific).

Techniques: Cell Culture, Control, Concentration Assay, Mineralization Assay, Staining

Journal: Acta Histochemica et Cytochemica

Article Title: Expression and Dynamics of Podoplanin in Cultured Osteoblasts with Mechanostress and Mineralization Stimulus

doi: 10.1267/ahc.17031

Figure Lengend Snippet: Immunostaining of osteoblasts cultured with anti-podplanin. The osteoblasts cultured in α-MEM without anti-podoplanin (left lane; 0 day, 0 μg/ml ) were immunostained by antibodies specific for podoplanin, osteopontin, and osteocalcin. There are little differences in staining intensities for podoplanin, and osteopontin between osteoblasts cultured in mineralization medium for 5 days (middle lane; 5 days, 0 μg/ml) and cells cultured in α-MEM without anti-podoplanin, while the intensity for osteocalcin increased. In osteoblasts cultured in mineralization medium for 5 days, the staining intensities for podoplanin, osteopontin, and osteocalcin are less in cells cultured with anti-podoplanin (middle lane; 5 days, 1 μg/ml) than in cells without anti-podoplanin (right lane; 5 days, 0 μg/ml). Nuclei were stained by DAPI. Bars = 100 μm.

Article Snippet: Contaminating genomic DNA was removed using DNAfree (Ambion, Huntingdon, UK), and the RT was performed on 30 ng of total RNA at 30°C for 10 min, 55°C for 30 min, and 99°C for 5 min, followed by 30 cycles of PCR for amplification (extension at 72°C for 30 sec, denaturation at 94°C for 30 sec, annealing at 60°C for 90 sec, and followed by final extension at 72°C for 10 min) using the Ex Taq hot start version (Takara Bio Inc., Otsu, Japan) with 50 pM of primer sets for mouse mRNA of β-actin (Mm02619580_g1; Thermo Fisher Scientific , Inc., Yokohama, JAPAN), podoplanin (Mm01348912_g1; Thermo Fisher Scientific), osteopontin (Mm00436767_m1; Thermo Fisher Scientific), and osteocalcin (Mm03413826_mH; Thermo Fisher Scientific), where the specificities had been confirmed by the manufacturer (Thermo Fisher Scientific).

Techniques: Immunostaining, Cell Culture, Staining

Journal: Acta Histochemica et Cytochemica

Article Title: Expression and Dynamics of Podoplanin in Cultured Osteoblasts with Mechanostress and Mineralization Stimulus

doi: 10.1267/ahc.17031

Figure Lengend Snippet: A. Ratio of immunostained areas in osteoblasts cultured with anti-podplanin. The relative amounts of the proteins were estimated by the ratio of the immunostained area. The osteoblasts cultured in α-MEM without anti-podoplanin were immunostained by podoplanin, osteopontin, and osteocalcin (0 day, 0 μg/ml). The staining intensities of podoplanin, osteopontin, and osteocalcin in osteoblasts cultured in mineralization medium for 5 days are statistically significantly weaker in cells with anti-podoplanin (5 days, 1 μg/ml) than in cells without anti-podoplanin (5 days, 0 μg/ml). In osteoblasts without anti-podoplanin, there are no significant differences in staining intensities for both podoplanin and osteopontin between cells in α-MEM (0 days, 0 μg/ml) and cells in mineralization medium (5 days, 0 μg/ml), while the staining intensity for osteocalcin is more in osteoblasts in mineralization medium (5 days, 0 μg/ml) than in cells in α-MEM (0 days, 0 μg/ml). *Significantly different in ANOVA (P < 0.01). B. Real time-PCR analysis for podoplanin, osteopontin, and osteocalcin mRNAs in osteoblasts cultured with anti-podplanin. The relative amounts of mRNAs were expressed by the means of the ratio (%): podoplanin, osteopontin, and osteocalcin cDNA units/β-actin cDNA units. The mRNA amounts of podoplanin, osteopontin, and osteocalcin in cells cultured with anti-podoplanin (5 days, 1 μg/ml) are statistically significantly smaller than in cells without anti-podoplanin (5 days, 0 μg/ml). In osteoblasts without anti-podoplanin, there are no significant differences in the mRNA amounts for both podoplanin and osteopontin between the cells in α-MEM (0 days, 0 μg/ml) and cells in mineralization medium (5 days, 0 μg/ml), while the mRNA amounts for osteocalcin is more in osteoblasts in mineralization medium (5 days, 0 μg/ml) than in cells in α-MEM (0 days, 0 μg/ml). Values are mean ± SD of data from 6 wells for each group. *Significantly different in ANOVA (P < 0.01).

Article Snippet: Contaminating genomic DNA was removed using DNAfree (Ambion, Huntingdon, UK), and the RT was performed on 30 ng of total RNA at 30°C for 10 min, 55°C for 30 min, and 99°C for 5 min, followed by 30 cycles of PCR for amplification (extension at 72°C for 30 sec, denaturation at 94°C for 30 sec, annealing at 60°C for 90 sec, and followed by final extension at 72°C for 10 min) using the Ex Taq hot start version (Takara Bio Inc., Otsu, Japan) with 50 pM of primer sets for mouse mRNA of β-actin (Mm02619580_g1; Thermo Fisher Scientific , Inc., Yokohama, JAPAN), podoplanin (Mm01348912_g1; Thermo Fisher Scientific), osteopontin (Mm00436767_m1; Thermo Fisher Scientific), and osteocalcin (Mm03413826_mH; Thermo Fisher Scientific), where the specificities had been confirmed by the manufacturer (Thermo Fisher Scientific).

Techniques: Cell Culture, Staining, Real-time Polymerase Chain Reaction

Journal: Journal of Clinical Medicine

Article Title: Comprehensive Gene Analysis of IgG4-Related Ophthalmic Disease Using RNA Sequencing

doi: 10.3390/jcm9113458

Figure Lengend Snippet: List of DEGs when the IgG4-ROD tissue was compared to all control tissues.

Article Snippet: Immunostaining by mouse anti-human MMP12 (1:50, MAB919, R&D Systems, Minneapolis, MN, USA) and rabbit anti-human SPP1 (1:1000, HPA027541, Atlas Antibodies, Bromma, Sweden) was performed using paraffin-embedded biopsy tissues.

Techniques: Control

Journal: Journal of Clinical Medicine

Article Title: Comprehensive Gene Analysis of IgG4-Related Ophthalmic Disease Using RNA Sequencing

doi: 10.3390/jcm9113458

Figure Lengend Snippet: Differentially expressed genes of IgG4-ROD versus orbital MALT lymphoma: top 40 in descending p value.

Article Snippet: Immunostaining by mouse anti-human MMP12 (1:50, MAB919, R&D Systems, Minneapolis, MN, USA) and rabbit anti-human SPP1 (1:1000, HPA027541, Atlas Antibodies, Bromma, Sweden) was performed using paraffin-embedded biopsy tissues.

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Comprehensive Gene Analysis of IgG4-Related Ophthalmic Disease Using RNA Sequencing

doi: 10.3390/jcm9113458

Figure Lengend Snippet: Differentially expressed genes of IgG4-ROD versus RLH: top 40 in descending p value.

Article Snippet: Immunostaining by mouse anti-human MMP12 (1:50, MAB919, R&D Systems, Minneapolis, MN, USA) and rabbit anti-human SPP1 (1:1000, HPA027541, Atlas Antibodies, Bromma, Sweden) was performed using paraffin-embedded biopsy tissues.

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Comprehensive Gene Analysis of IgG4-Related Ophthalmic Disease Using RNA Sequencing

doi: 10.3390/jcm9113458

Figure Lengend Snippet: Results of real-time PCR for validation of differentially expressed genes obtained from RNA sequencing. *** Messenger RNA of MMP12 and SPP1 in immunoglobulin G4-related ophthalmic disease is significantly higher than mucosa-associated lymphoid tissue lymphoma ( p = 2.7141 × 10 −8 and 1.2044 × 10 −7 , respectively). Statistical analysis was performed by a Mann–Whitney U test ( n = 30). IgG4-ROD: Immunoglobulin G4 related ophthalmic disease, MALT: Mucosa-associated lymph tissue, MMP12: Matrix metallopeptidase 12, SPP1: Secreted phosphoprotein 1.

Article Snippet: Immunostaining by mouse anti-human MMP12 (1:50, MAB919, R&D Systems, Minneapolis, MN, USA) and rabbit anti-human SPP1 (1:1000, HPA027541, Atlas Antibodies, Bromma, Sweden) was performed using paraffin-embedded biopsy tissues.

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, RNA Sequencing, MANN-WHITNEY

Journal: Journal of Clinical Medicine

Article Title: Comprehensive Gene Analysis of IgG4-Related Ophthalmic Disease Using RNA Sequencing

doi: 10.3390/jcm9113458

Figure Lengend Snippet: Representative immunohistochemical staining patterns of formalin-fixed, paraffin-embedded sections (MMP12: magnification 400×, bar: 50 μm, SPP1: magnification 200×, bar: 100μm). The upper row is immunoreactivity of MMP12, and the lower row is immunoreactivity of SPP1. From left column to right column, IgG4-ROD, MALT lymphoma, RLH, and the lacrimal gland tissue section are shown. Many MMP12 positive cells are observable in the fibrotic areas and follicular areas of IgG4-ROD tissues. SPP1 is intensely stained in the fibrotic part of IgG4-ROD. IgG4-ROD: Immunoglobulin G4 related ophthalmic disease, MALT: Mucosa-associated lymph tissue, RLH: Reactive lymphoid hyperplasia, MMP12: Matrix metallopeptidase 12, SPP1: Secreted phosphoprotein 1.

Article Snippet: Immunostaining by mouse anti-human MMP12 (1:50, MAB919, R&D Systems, Minneapolis, MN, USA) and rabbit anti-human SPP1 (1:1000, HPA027541, Atlas Antibodies, Bromma, Sweden) was performed using paraffin-embedded biopsy tissues.

Techniques: Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded