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    Structured Review

    Millipore β glycerophosphate
    Evaluation of MC3T3 cell differentiation. MC3T3 cells were differentiated for 2, 15 or 29 days in the presence of 50 μg/ml L-ascorbic acid and 10 mM <t>β-glycerophosphate.</t> Then, matrix mineralization was evaluated using alizarin red staining ( A ), and LCN2 ( B ), ALP ( C ), Runx2 ( D ) and osteocalcin ( E ) gene expression was evaluated by real-time PCR. MC3T3 cells were differentiated for 15 days as described above and then were treated for 48 h with the de-differentiating factor 10 μM dexamethasone (Dx) to measure LCN2 ( F ) and ALP ( G ) gene expression. The results are presented as the mean ± SEM of at least three independent experiments. *p
    β Glycerophosphate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 256 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β glycerophosphate/product/Millipore
    Average 99 stars, based on 256 article reviews
    Price from $9.99 to $1999.99
    β glycerophosphate - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "The adipokine lipocalin-2 in the context of the osteoarthritic osteochondral junction"

    Article Title: The adipokine lipocalin-2 in the context of the osteoarthritic osteochondral junction

    Journal: Scientific Reports

    doi: 10.1038/srep29243

    Evaluation of MC3T3 cell differentiation. MC3T3 cells were differentiated for 2, 15 or 29 days in the presence of 50 μg/ml L-ascorbic acid and 10 mM β-glycerophosphate. Then, matrix mineralization was evaluated using alizarin red staining ( A ), and LCN2 ( B ), ALP ( C ), Runx2 ( D ) and osteocalcin ( E ) gene expression was evaluated by real-time PCR. MC3T3 cells were differentiated for 15 days as described above and then were treated for 48 h with the de-differentiating factor 10 μM dexamethasone (Dx) to measure LCN2 ( F ) and ALP ( G ) gene expression. The results are presented as the mean ± SEM of at least three independent experiments. *p
    Figure Legend Snippet: Evaluation of MC3T3 cell differentiation. MC3T3 cells were differentiated for 2, 15 or 29 days in the presence of 50 μg/ml L-ascorbic acid and 10 mM β-glycerophosphate. Then, matrix mineralization was evaluated using alizarin red staining ( A ), and LCN2 ( B ), ALP ( C ), Runx2 ( D ) and osteocalcin ( E ) gene expression was evaluated by real-time PCR. MC3T3 cells were differentiated for 15 days as described above and then were treated for 48 h with the de-differentiating factor 10 μM dexamethasone (Dx) to measure LCN2 ( F ) and ALP ( G ) gene expression. The results are presented as the mean ± SEM of at least three independent experiments. *p

    Techniques Used: Cell Differentiation, Staining, ALP Assay, Expressing, Real-time Polymerase Chain Reaction

    2) Product Images from "The protective effects of Achyranthes bidentata root extract on the antimycin A induced damage of osteoblastic MC3T3-E1 cells"

    Article Title: The protective effects of Achyranthes bidentata root extract on the antimycin A induced damage of osteoblastic MC3T3-E1 cells

    Journal: Cytotechnology

    doi: 10.1007/s10616-013-9645-4

    Effect of A. bidentata extract (AE) on the cell viability and mineralization of osteoblasts in the presence of antimycin A (AMA). The cells were treated, at confluence, with culture medium containing 5 mM β-glycerophosphate and 50 μg/ml
    Figure Legend Snippet: Effect of A. bidentata extract (AE) on the cell viability and mineralization of osteoblasts in the presence of antimycin A (AMA). The cells were treated, at confluence, with culture medium containing 5 mM β-glycerophosphate and 50 μg/ml

    Techniques Used:

    3) Product Images from "NOD2 Mediates Odontoblast Differentiation and RANKL Expression"

    Article Title: NOD2 Mediates Odontoblast Differentiation and RANKL Expression

    Journal: Journal of Dental Research

    doi: 10.1177/0022034514535214

    MDP treatment negatively regulates odontoblast differentiation through NOD2 in HDPCs. (A) HDPCs were cultured with MDP at concentrations ranging from 0 to 20 µg/mL for the indicated days in osteogenic media (10 mM β-glycerophosphate, 10
    Figure Legend Snippet: MDP treatment negatively regulates odontoblast differentiation through NOD2 in HDPCs. (A) HDPCs were cultured with MDP at concentrations ranging from 0 to 20 µg/mL for the indicated days in osteogenic media (10 mM β-glycerophosphate, 10

    Techniques Used: Cell Culture

    MDP treatment reduced phosphorylation of MAPKs via the induction of MAPK phosphatase (MKP)-1 in HDPCs. (A) HDPCs were stimulated with MDP at 10 µg/mL for 120 min in osteogenic media (OM) with 10 mM β-glycerophosphate, 10 −7 M dexamethasone,
    Figure Legend Snippet: MDP treatment reduced phosphorylation of MAPKs via the induction of MAPK phosphatase (MKP)-1 in HDPCs. (A) HDPCs were stimulated with MDP at 10 µg/mL for 120 min in osteogenic media (OM) with 10 mM β-glycerophosphate, 10 −7 M dexamethasone,

    Techniques Used:

    4) Product Images from "Adiponectin Deficiency Triggers Bone Loss by Up-Regulation of Osteoclastogenesis and Down-Regulation of Osteoblastogenesis"

    Article Title: Adiponectin Deficiency Triggers Bone Loss by Up-Regulation of Osteoclastogenesis and Down-Regulation of Osteoblastogenesis

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2019.00815

    Adiponectin-deficient calvarial cells preferentially differentiate into adipocytes. (A–C) Mouse calvarial cells were incubated with β-glycerophosphate (10 mM) and ascorbic acid (50 μM) to induce osteoblast differentiation. (A) The cells were stained for ALP at day 7 and with alizarin red S at day 21 (upper, whole-well image; lower, 40× magnification). (B) Alizarin red S-stained cells were dissolved in 20% methanol and 10% acetic acid and the optical density (OD) was measured at 450 nm. (C) At days 5 and 10, total RNAs were isolated, reverse-transcribed, and subjected to RT-PCR to determine the mRNA levels of BSP, OCN, ALP, β-catenin, Runx2, and β-actin. (D–F) Mouse calvarial cells were incubated with dexamethasone (1 μM), insulin (5 μg/ml), and IBMX (0.5 mM) for the evaluation of adipogenic potential. (D) At day 21, the cells were fixed and stained using oil red O (upper, whole-well image; lower, 40× magnification). (E) The oil red O-stained cells were dissolved in isopropanol and the OD was measured at 500 nm. (F) At day 5, total RNAs were isolated, reverse-transcribed, and subjected to RT-PCR. The ratio of PPARγ to β-actin was obtained by using a densitometer. Data are mean values ± SD of triplicate samples and are representative of three similar independent experiments. * P
    Figure Legend Snippet: Adiponectin-deficient calvarial cells preferentially differentiate into adipocytes. (A–C) Mouse calvarial cells were incubated with β-glycerophosphate (10 mM) and ascorbic acid (50 μM) to induce osteoblast differentiation. (A) The cells were stained for ALP at day 7 and with alizarin red S at day 21 (upper, whole-well image; lower, 40× magnification). (B) Alizarin red S-stained cells were dissolved in 20% methanol and 10% acetic acid and the optical density (OD) was measured at 450 nm. (C) At days 5 and 10, total RNAs were isolated, reverse-transcribed, and subjected to RT-PCR to determine the mRNA levels of BSP, OCN, ALP, β-catenin, Runx2, and β-actin. (D–F) Mouse calvarial cells were incubated with dexamethasone (1 μM), insulin (5 μg/ml), and IBMX (0.5 mM) for the evaluation of adipogenic potential. (D) At day 21, the cells were fixed and stained using oil red O (upper, whole-well image; lower, 40× magnification). (E) The oil red O-stained cells were dissolved in isopropanol and the OD was measured at 500 nm. (F) At day 5, total RNAs were isolated, reverse-transcribed, and subjected to RT-PCR. The ratio of PPARγ to β-actin was obtained by using a densitometer. Data are mean values ± SD of triplicate samples and are representative of three similar independent experiments. * P

    Techniques Used: Incubation, Staining, ALP Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    5) Product Images from "Kruppel-like factor 4 attenuates osteoblast formation, function, and cross talk with osteoclasts"

    Article Title: Kruppel-like factor 4 attenuates osteoblast formation, function, and cross talk with osteoclasts

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201308102

    Expression of KLF4 during osteoblast differentiation. Osteoblasts were cultured with osteogenic medium containing BMP2 (100 ng/ml), ascorbic acid (50 µg/ml), and β-glycerophosphate (100 mM) for the indicated times. Quantitative real-time PCR was performed for the mRNA expression of Runx2 , Alpl (AP), Ibsp (BSP), and Klf4 . Data represent means ± SD of triplicate samples.
    Figure Legend Snippet: Expression of KLF4 during osteoblast differentiation. Osteoblasts were cultured with osteogenic medium containing BMP2 (100 ng/ml), ascorbic acid (50 µg/ml), and β-glycerophosphate (100 mM) for the indicated times. Quantitative real-time PCR was performed for the mRNA expression of Runx2 , Alpl (AP), Ibsp (BSP), and Klf4 . Data represent means ± SD of triplicate samples.

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    Expression of KLF4 in osteoclasts and osteoblasts. (A) Total RNA was isolated from bone, spleen, brain, thymus, lung, kidney, liver, intestine, heart, and muscle of mice. To obtain osteoclasts, BMMs were cultured with M-CSF (30 ng/ml) and RANKL (150 ng/ml) for 3 d. To obtain osteoblasts, primary osteoblast precursor cells were cultured with osteogenic medium containing BMP2 (100 ng/ml), ascorbic acid (50 µg/ml), and β-glycerophosphate (100 mM) for 6 d. Quantitative real-time PCR was performed for the mRNA expression of Klf4 . (B) Primary calvarial osteoblasts were cultured with 1,25(OH) 2 D 3 (10 nM) for the indicated times. mRNA expression of Tnfsf11 (RANKL), Tnfrsf11b (OPG), and Klf4 was measured by quantitative real-time PCR. (C) For osteoclast differentiation, BMMs were cultured with M-CSF (30 ng/ml) and RANKL (150 ng/ml) for the indicated times. mRNA levels of Nfatc1 , Acp5 (TRAP), and Klf4 were assessed by quantitative real-time PCR. Data represent means ± SD of triplicate samples.
    Figure Legend Snippet: Expression of KLF4 in osteoclasts and osteoblasts. (A) Total RNA was isolated from bone, spleen, brain, thymus, lung, kidney, liver, intestine, heart, and muscle of mice. To obtain osteoclasts, BMMs were cultured with M-CSF (30 ng/ml) and RANKL (150 ng/ml) for 3 d. To obtain osteoblasts, primary osteoblast precursor cells were cultured with osteogenic medium containing BMP2 (100 ng/ml), ascorbic acid (50 µg/ml), and β-glycerophosphate (100 mM) for 6 d. Quantitative real-time PCR was performed for the mRNA expression of Klf4 . (B) Primary calvarial osteoblasts were cultured with 1,25(OH) 2 D 3 (10 nM) for the indicated times. mRNA expression of Tnfsf11 (RANKL), Tnfrsf11b (OPG), and Klf4 was measured by quantitative real-time PCR. (C) For osteoclast differentiation, BMMs were cultured with M-CSF (30 ng/ml) and RANKL (150 ng/ml) for the indicated times. mRNA levels of Nfatc1 , Acp5 (TRAP), and Klf4 were assessed by quantitative real-time PCR. Data represent means ± SD of triplicate samples.

    Techniques Used: Expressing, Isolation, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction

    6) Product Images from "Osteoblastogenesis from synovial fluid-derived cells is related to the type and severity of juvenile idiopathic arthritis"

    Article Title: Osteoblastogenesis from synovial fluid-derived cells is related to the type and severity of juvenile idiopathic arthritis

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar3872

    Effect of synovial fluid from patients with oligoarticular form of juvenile idiopathic arthritis (oJIA) and polyarticular form of JIA (pJIA) on the differentiation of osteoblasts from normal human bone marrow (BM) progenitors . Osteoblastogenesis was induced by ascorbic acid and β-glycerophosphate. Synovial fluid from patients with oJIA or pJIA was added to osteoblastogenic culture with each medium exchange. Osteoblastogenesis was assessed by AP activity and expression of Runx2 gene by real time PCR. (A) AP activity measured at day 7 and 17 in control cultures (hBM) and cultures supplemented with 10% synovial fluid from children with oJIA (hBM + oJIA SF) and pJIA (hBM + oJIA SF, * P
    Figure Legend Snippet: Effect of synovial fluid from patients with oligoarticular form of juvenile idiopathic arthritis (oJIA) and polyarticular form of JIA (pJIA) on the differentiation of osteoblasts from normal human bone marrow (BM) progenitors . Osteoblastogenesis was induced by ascorbic acid and β-glycerophosphate. Synovial fluid from patients with oJIA or pJIA was added to osteoblastogenic culture with each medium exchange. Osteoblastogenesis was assessed by AP activity and expression of Runx2 gene by real time PCR. (A) AP activity measured at day 7 and 17 in control cultures (hBM) and cultures supplemented with 10% synovial fluid from children with oJIA (hBM + oJIA SF) and pJIA (hBM + oJIA SF, * P

    Techniques Used: Activity Assay, Expressing, Real-time Polymerase Chain Reaction

    Differentiation of osteoblasts from primary synovial fluid (SF)-derived cells and SF-derived fibroblasts from the fourth passage (P4), from patients with oligoarticular juvenile idiopathic arthritis (oJIA) and polyarticular JIA (pJIA) . Osteoblast differentiation was induced by ascorbic acid and β-glycerophosphate, and assessed by alkaline phosphatase (AP) histochemical staining. Total fibroblastic colonies were visualized with methylene blue (MB). (A) Osteoblast and total colonies from representative primary SF-derived cells from children with oJIA and pJIA stained for AP and MB on culture day 21. (B) AP-stained area (median and interquartile range (IQR), lines represent minimum and maximum values) in osteoblast cultures grown from primary SF-derived cells from children with oJIA (n = 18) and pJIA (n = 9); * P
    Figure Legend Snippet: Differentiation of osteoblasts from primary synovial fluid (SF)-derived cells and SF-derived fibroblasts from the fourth passage (P4), from patients with oligoarticular juvenile idiopathic arthritis (oJIA) and polyarticular JIA (pJIA) . Osteoblast differentiation was induced by ascorbic acid and β-glycerophosphate, and assessed by alkaline phosphatase (AP) histochemical staining. Total fibroblastic colonies were visualized with methylene blue (MB). (A) Osteoblast and total colonies from representative primary SF-derived cells from children with oJIA and pJIA stained for AP and MB on culture day 21. (B) AP-stained area (median and interquartile range (IQR), lines represent minimum and maximum values) in osteoblast cultures grown from primary SF-derived cells from children with oJIA (n = 18) and pJIA (n = 9); * P

    Techniques Used: Derivative Assay, Staining

    7) Product Images from "Propofol attenuates osteoclastogenesis by lowering RANKL/OPG ratio in mouse osteoblasts"

    Article Title: Propofol attenuates osteoclastogenesis by lowering RANKL/OPG ratio in mouse osteoblasts

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.22713

    Propofol does not affect osteoblast differentiation. (A) Calvarial pre-osteoblast cells isolated from newborn mice were cultured in osteogenic media (OM, 10 mM β-glycerophosphate + 100 μM L-ascorbic acid) for the indicated number of days. At day 4 and day 8, osteoblast differentiation was examined by ALP staining. (B) Calvarial pre-osteoblast cells were differentiated into osteoblast in OM. At days 0, 4, and 8, quantitative ALP enzyme activity assay was performed.
    Figure Legend Snippet: Propofol does not affect osteoblast differentiation. (A) Calvarial pre-osteoblast cells isolated from newborn mice were cultured in osteogenic media (OM, 10 mM β-glycerophosphate + 100 μM L-ascorbic acid) for the indicated number of days. At day 4 and day 8, osteoblast differentiation was examined by ALP staining. (B) Calvarial pre-osteoblast cells were differentiated into osteoblast in OM. At days 0, 4, and 8, quantitative ALP enzyme activity assay was performed.

    Techniques Used: Isolation, Mouse Assay, Cell Culture, ALP Assay, Staining, Enzyme Activity Assay

    Propofol does not exert cytotoxic effects or alter cell proliferation in primary calvarial osteoblasts. (A) Calvarial osteoblasts were incubated in medium containing indicated concentrations of propofol (0-100 μM) for 24 hours. Cell viability was evaluated by MTT assay. (B) Calvarial osteoblasts were cultured in osteogenic media (OM, 10 mM β-glycerophosphate + 100 μM L-ascorbic acid) for 3 days in the presence of indicated doses of propofol (0-100 μM). Cell proliferation was measured at daily intervals by MTT assay.
    Figure Legend Snippet: Propofol does not exert cytotoxic effects or alter cell proliferation in primary calvarial osteoblasts. (A) Calvarial osteoblasts were incubated in medium containing indicated concentrations of propofol (0-100 μM) for 24 hours. Cell viability was evaluated by MTT assay. (B) Calvarial osteoblasts were cultured in osteogenic media (OM, 10 mM β-glycerophosphate + 100 μM L-ascorbic acid) for 3 days in the presence of indicated doses of propofol (0-100 μM). Cell proliferation was measured at daily intervals by MTT assay.

    Techniques Used: Incubation, MTT Assay, Cell Culture

    8) Product Images from "Rev-erbα Negatively Regulates Osteoclast and Osteoblast Differentiation through p38 MAPK Signaling Pathway"

    Article Title: Rev-erbα Negatively Regulates Osteoclast and Osteoblast Differentiation through p38 MAPK Signaling Pathway

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2019.0232

    Expression of Rev-erbs during osteoblast differentiation and their effect on osteoblast differentiation BMSCs were incubated with osteogenic medium containing IGF-1 (50 ng/ml), ascorbic acid (50 μg/ml), and β-glycerophosphate (100 μM) for the indicated times. (A) Total RNA was isolated from the cell lysates and real-time PCR was performed to determine mRNA expression of Rev-erb α, Rev-erb β, Alpl , Runx2 , and Bglap . The data represent mean ± SD of triplicate samples. The values shown are normalized to GAPDH levels. # P
    Figure Legend Snippet: Expression of Rev-erbs during osteoblast differentiation and their effect on osteoblast differentiation BMSCs were incubated with osteogenic medium containing IGF-1 (50 ng/ml), ascorbic acid (50 μg/ml), and β-glycerophosphate (100 μM) for the indicated times. (A) Total RNA was isolated from the cell lysates and real-time PCR was performed to determine mRNA expression of Rev-erb α, Rev-erb β, Alpl , Runx2 , and Bglap . The data represent mean ± SD of triplicate samples. The values shown are normalized to GAPDH levels. # P

    Techniques Used: Expressing, Incubation, Isolation, Real-time Polymerase Chain Reaction

    Rev-erbα overexpression inhibits osteoblast differentiation via regulation of Runx2 nuclear translocation BMSCs were transduced with either pMX-IRES-EGFP (control) or Rev-erbα retroviruses, and cultured with osteogenic medium containing IGF-1, ascorbic acid, and β-glycerophosphate. (A) Cells were cultured for 4 days, and ALP activities were measured by densitometry at 405 nm. O.D., optical density. The data represent mean ± SD of triplicate samples. * P
    Figure Legend Snippet: Rev-erbα overexpression inhibits osteoblast differentiation via regulation of Runx2 nuclear translocation BMSCs were transduced with either pMX-IRES-EGFP (control) or Rev-erbα retroviruses, and cultured with osteogenic medium containing IGF-1, ascorbic acid, and β-glycerophosphate. (A) Cells were cultured for 4 days, and ALP activities were measured by densitometry at 405 nm. O.D., optical density. The data represent mean ± SD of triplicate samples. * P

    Techniques Used: Over Expression, Translocation Assay, Transduction, Cell Culture

    9) Product Images from "Propofol attenuates osteoclastogenesis by lowering RANKL/OPG ratio in mouse osteoblasts"

    Article Title: Propofol attenuates osteoclastogenesis by lowering RANKL/OPG ratio in mouse osteoblasts

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.22713

    Propofol does not affect osteoblast differentiation. (A) Calvarial pre-osteoblast cells isolated from newborn mice were cultured in osteogenic media (OM, 10 mM β-glycerophosphate + 100 μM L-ascorbic acid) for the indicated number of days. At day 4 and day 8, osteoblast differentiation was examined by ALP staining. (B) Calvarial pre-osteoblast cells were differentiated into osteoblast in OM. At days 0, 4, and 8, quantitative ALP enzyme activity assay was performed.
    Figure Legend Snippet: Propofol does not affect osteoblast differentiation. (A) Calvarial pre-osteoblast cells isolated from newborn mice were cultured in osteogenic media (OM, 10 mM β-glycerophosphate + 100 μM L-ascorbic acid) for the indicated number of days. At day 4 and day 8, osteoblast differentiation was examined by ALP staining. (B) Calvarial pre-osteoblast cells were differentiated into osteoblast in OM. At days 0, 4, and 8, quantitative ALP enzyme activity assay was performed.

    Techniques Used: Isolation, Mouse Assay, Cell Culture, ALP Assay, Staining, Enzyme Activity Assay

    Propofol does not exert cytotoxic effects or alter cell proliferation in primary calvarial osteoblasts. (A) Calvarial osteoblasts were incubated in medium containing indicated concentrations of propofol (0-100 μM) for 24 hours. Cell viability was evaluated by MTT assay. (B) Calvarial osteoblasts were cultured in osteogenic media (OM, 10 mM β-glycerophosphate + 100 μM L-ascorbic acid) for 3 days in the presence of indicated doses of propofol (0-100 μM). Cell proliferation was measured at daily intervals by MTT assay.
    Figure Legend Snippet: Propofol does not exert cytotoxic effects or alter cell proliferation in primary calvarial osteoblasts. (A) Calvarial osteoblasts were incubated in medium containing indicated concentrations of propofol (0-100 μM) for 24 hours. Cell viability was evaluated by MTT assay. (B) Calvarial osteoblasts were cultured in osteogenic media (OM, 10 mM β-glycerophosphate + 100 μM L-ascorbic acid) for 3 days in the presence of indicated doses of propofol (0-100 μM). Cell proliferation was measured at daily intervals by MTT assay.

    Techniques Used: Incubation, MTT Assay, Cell Culture

    10) Product Images from "Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions"

    Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23453

    Effects of estradiol on osteoblast maturation Primary rat osteoblasts isolated from neonatal calvarias were exposed to a combination of estradiol (10 nM) and the differentiation agent, including dexamethasone, ascorbic acid, and β-glycerophosphate, for 21 days. Control cells received the differentiation agent only. Cell morphology was observed using a light microscope (A) . The symbol, →, indicates a calcified nodule. Alkaline phosphatase (ALP) activity was assayed with a colorimetric method (B) . Mineralized nodules were stained using Alizarin red S- (C) and the von Kossa-staining (E) protocols. These nodule signals were quantified and statistically analyzed (D and F) . Each value represent the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p
    Figure Legend Snippet: Effects of estradiol on osteoblast maturation Primary rat osteoblasts isolated from neonatal calvarias were exposed to a combination of estradiol (10 nM) and the differentiation agent, including dexamethasone, ascorbic acid, and β-glycerophosphate, for 21 days. Control cells received the differentiation agent only. Cell morphology was observed using a light microscope (A) . The symbol, →, indicates a calcified nodule. Alkaline phosphatase (ALP) activity was assayed with a colorimetric method (B) . Mineralized nodules were stained using Alizarin red S- (C) and the von Kossa-staining (E) protocols. These nodule signals were quantified and statistically analyzed (D and F) . Each value represent the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p

    Techniques Used: Isolation, Light Microscopy, ALP Assay, Activity Assay, Staining

    11) Product Images from "SGK1 induces vascular smooth muscle cell calcification through NF- κB signaling"

    Article Title: SGK1 induces vascular smooth muscle cell calcification through NF- κB signaling

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI96477

    SGK1 inhibition ameliorates phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary HAoSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 7 per group; AU) of SGK1 relative mRNA expression in HAoSMCs following treatment with control or β-glycerophosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). ( B ) Representative confocal microscopy images ( n = 3 per group) showing NF-κB p65 protein expression and localization in HAoSMCs following treatment with control or β-glycerophosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). Green labeling, NF-κB p65 expression; magenta labeling, nuclei. Scale bars: 20 μm. ( C ) Representative original images ( n = 4 per group) showing alizarin red staining in HAoSMCs following treatment with control or with calcification medium without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). Calcified areas are shown as red staining. ( D – H ) Scatter dot plots and arithmetic means ± SEM of calcium content ( D , n = 6 per group, μg/mg protein), alkaline phosphatase activity ( E , n = 4 per group, U/mg protein), and MSX2 ( F ), CBFA1 ( G ), and ALPL ( H ) relative mRNA expression ( n = 7 per group; AU) in HAoSMCs following treatment with control or phosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). * P
    Figure Legend Snippet: SGK1 inhibition ameliorates phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary HAoSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 7 per group; AU) of SGK1 relative mRNA expression in HAoSMCs following treatment with control or β-glycerophosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). ( B ) Representative confocal microscopy images ( n = 3 per group) showing NF-κB p65 protein expression and localization in HAoSMCs following treatment with control or β-glycerophosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). Green labeling, NF-κB p65 expression; magenta labeling, nuclei. Scale bars: 20 μm. ( C ) Representative original images ( n = 4 per group) showing alizarin red staining in HAoSMCs following treatment with control or with calcification medium without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). Calcified areas are shown as red staining. ( D – H ) Scatter dot plots and arithmetic means ± SEM of calcium content ( D , n = 6 per group, μg/mg protein), alkaline phosphatase activity ( E , n = 4 per group, U/mg protein), and MSX2 ( F ), CBFA1 ( G ), and ALPL ( H ) relative mRNA expression ( n = 7 per group; AU) in HAoSMCs following treatment with control or phosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). * P

    Techniques Used: Inhibition, Expressing, Confocal Microscopy, Labeling, Staining, Activity Assay

    Sgk1 deficiency reduces phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary MAoSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 6 per group; AU) of Sgk1 relative mRNA expression in primary MAoSMCs isolated from WT mice (sgk1 +/+ ) and treated with control or β-glycerophosphate (Pi). ( B ) Representative confocal microscopy images ( n = 3 per group) showing Sgk1 and NF-κB p65 protein expression and scatter dot plots and arithmetic means ± SEM ( n = 3 per group; AU) of normalized Sgk1 fluorescence intensity in sgk1 –/– or sgk1 +/+ MAoSMCs treated with control or Pi. Green labeling, Sgk1 or NF-κB p65 expression; magenta labeling, nuclei. Scale bars: 20 μm. ** P
    Figure Legend Snippet: Sgk1 deficiency reduces phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary MAoSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 6 per group; AU) of Sgk1 relative mRNA expression in primary MAoSMCs isolated from WT mice (sgk1 +/+ ) and treated with control or β-glycerophosphate (Pi). ( B ) Representative confocal microscopy images ( n = 3 per group) showing Sgk1 and NF-κB p65 protein expression and scatter dot plots and arithmetic means ± SEM ( n = 3 per group; AU) of normalized Sgk1 fluorescence intensity in sgk1 –/– or sgk1 +/+ MAoSMCs treated with control or Pi. Green labeling, Sgk1 or NF-κB p65 expression; magenta labeling, nuclei. Scale bars: 20 μm. ** P

    Techniques Used: Expressing, Isolation, Mouse Assay, Confocal Microscopy, Fluorescence, Labeling

    SGK1 expression in VSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 6 per group; arbitrary units [AU]) of SGK1 relative mRNA expression in primary HAoSMCs following treatment with control (CTR) or aldosterone (Aldo), dexamethasone (Dex), β-glycerophosphate (Pi), glucose, recombinant human TGF-β1 protein, or recombinant human BMP-2 protein. ( B ) Representative original Western blots and scatter dot plots and arithmetic means ± SEM ( n = 4 per group; AU) of normalized SGK1/GAPDH protein ratio in HAoSMCs following treatment with triggers of osteo-chondrogenic transdifferentiation. * P
    Figure Legend Snippet: SGK1 expression in VSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 6 per group; arbitrary units [AU]) of SGK1 relative mRNA expression in primary HAoSMCs following treatment with control (CTR) or aldosterone (Aldo), dexamethasone (Dex), β-glycerophosphate (Pi), glucose, recombinant human TGF-β1 protein, or recombinant human BMP-2 protein. ( B ) Representative original Western blots and scatter dot plots and arithmetic means ± SEM ( n = 4 per group; AU) of normalized SGK1/GAPDH protein ratio in HAoSMCs following treatment with triggers of osteo-chondrogenic transdifferentiation. * P

    Techniques Used: Expressing, Recombinant, Western Blot

    12) Product Images from "Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin"

    Article Title: Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin

    Journal: Human Cell

    doi: 10.1007/s13577-018-0202-5

    Measurements of cell proliferation by alamar blue assay on DPSC ( a ), Saos-2 ( b ) and HEPM ( c ) cells cultured in control medium (CM) supplemented with different components of the OIM medium such as: ascorbic acid (AA), β-glycerophosphate (β-GLY), dexamethasone (DEX), vitamin D3 (D3 VIT). Those components which decreased proliferation (β-glycerophosphate and dexamethasone) were combined with BMP-2 and were examined on DPSC ( d ), Saos-2 ( e ) and HEPM ( f ) cells. Error bars represent standard deviation calculated from three parallel measurements. Statistical analysis was performed using ANOVA followed by Bonferroni statistical test. P
    Figure Legend Snippet: Measurements of cell proliferation by alamar blue assay on DPSC ( a ), Saos-2 ( b ) and HEPM ( c ) cells cultured in control medium (CM) supplemented with different components of the OIM medium such as: ascorbic acid (AA), β-glycerophosphate (β-GLY), dexamethasone (DEX), vitamin D3 (D3 VIT). Those components which decreased proliferation (β-glycerophosphate and dexamethasone) were combined with BMP-2 and were examined on DPSC ( d ), Saos-2 ( e ) and HEPM ( f ) cells. Error bars represent standard deviation calculated from three parallel measurements. Statistical analysis was performed using ANOVA followed by Bonferroni statistical test. P

    Techniques Used: Alamar Blue Assay, Cell Culture, Standard Deviation

    13) Product Images from "Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin"

    Article Title: Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin

    Journal: Human Cell

    doi: 10.1007/s13577-018-0202-5

    Measurements of cell proliferation by alamar blue assay on DPSC ( a ), Saos-2 ( b ) and HEPM ( c ) cells cultured in control medium (CM) supplemented with different components of the OIM medium such as: ascorbic acid (AA), β-glycerophosphate (β-GLY), dexamethasone (DEX), vitamin D3 (D3 VIT). Those components which decreased proliferation (β-glycerophosphate and dexamethasone) were combined with BMP-2 and were examined on DPSC ( d ), Saos-2 ( e ) and HEPM ( f ) cells. Error bars represent standard deviation calculated from three parallel measurements. Statistical analysis was performed using ANOVA followed by Bonferroni statistical test. P
    Figure Legend Snippet: Measurements of cell proliferation by alamar blue assay on DPSC ( a ), Saos-2 ( b ) and HEPM ( c ) cells cultured in control medium (CM) supplemented with different components of the OIM medium such as: ascorbic acid (AA), β-glycerophosphate (β-GLY), dexamethasone (DEX), vitamin D3 (D3 VIT). Those components which decreased proliferation (β-glycerophosphate and dexamethasone) were combined with BMP-2 and were examined on DPSC ( d ), Saos-2 ( e ) and HEPM ( f ) cells. Error bars represent standard deviation calculated from three parallel measurements. Statistical analysis was performed using ANOVA followed by Bonferroni statistical test. P

    Techniques Used: Alamar Blue Assay, Cell Culture, Standard Deviation

    14) Product Images from "Calcium Supplement by Tetracycline guided amorphous Calcium Carbonate potentiates Osteoblast promotion for Synergetic Osteoporosis Therapy"

    Article Title: Calcium Supplement by Tetracycline guided amorphous Calcium Carbonate potentiates Osteoblast promotion for Synergetic Osteoporosis Therapy

    Journal: Theranostics

    doi: 10.7150/thno.45142

    Effects of Sim-loaded nanoparticles on mineralized nodule formation and ALP activity in MC3T3-E1 cells. MC3T3-E1 cells were cultured in standard osteogenic differentiation and mineralization medium containing 10 mM β-glycerophosphate, 50 µg/mL ascorbic acid and different formulations at the equal Sim concentration of 10 -7 M). Afterwards, the mineralization of the extracellular matrix was evaluated by optical microscopy and macroscopic observation ( A ), quantitative mineralization results ( B ) at 14 days post incubation, and stimulation of ALP activity ( C ) at 7 days post incubation. Results were expressed as mean ± S.D. ( n = 3). *p
    Figure Legend Snippet: Effects of Sim-loaded nanoparticles on mineralized nodule formation and ALP activity in MC3T3-E1 cells. MC3T3-E1 cells were cultured in standard osteogenic differentiation and mineralization medium containing 10 mM β-glycerophosphate, 50 µg/mL ascorbic acid and different formulations at the equal Sim concentration of 10 -7 M). Afterwards, the mineralization of the extracellular matrix was evaluated by optical microscopy and macroscopic observation ( A ), quantitative mineralization results ( B ) at 14 days post incubation, and stimulation of ALP activity ( C ) at 7 days post incubation. Results were expressed as mean ± S.D. ( n = 3). *p

    Techniques Used: Activity Assay, Cell Culture, Concentration Assay, Microscopy, Incubation

    15) Product Images from "miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis"

    Article Title: miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis

    Journal: Oncotarget

    doi:

    microRNA levels are altered during osteogenesis Osteoblast differentiation was induced in MC3T3 cells with dexamethasone, β-glycerophosphate and ascorbic acid. A. ALP staining detected ALP activity in cells grown with osteogenic differentiation but not with basal media (without osteogenic differentiation supplements); Alizarin Red S staining detected presence of calcium deposits (mineralization) in cells grown in osteogenic differentiation media but not in basal media (10X; scale: 100 μm). B. ALP, RUNX2, OSX and OPN mRNA levels were measured by quantitative real-time PCR. GAPDH was used as reference control. Expression levels at day 1 (D1), day 3 (D3), day 7 (D7) and day 14 (D14) of differentiation (dif) were normalized to expression levels of cells grown in basal conditions for the same time points (mean±SD, N = 4; * P
    Figure Legend Snippet: microRNA levels are altered during osteogenesis Osteoblast differentiation was induced in MC3T3 cells with dexamethasone, β-glycerophosphate and ascorbic acid. A. ALP staining detected ALP activity in cells grown with osteogenic differentiation but not with basal media (without osteogenic differentiation supplements); Alizarin Red S staining detected presence of calcium deposits (mineralization) in cells grown in osteogenic differentiation media but not in basal media (10X; scale: 100 μm). B. ALP, RUNX2, OSX and OPN mRNA levels were measured by quantitative real-time PCR. GAPDH was used as reference control. Expression levels at day 1 (D1), day 3 (D3), day 7 (D7) and day 14 (D14) of differentiation (dif) were normalized to expression levels of cells grown in basal conditions for the same time points (mean±SD, N = 4; * P

    Techniques Used: ALP Assay, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Expressing

    16) Product Images from "Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment"

    Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

    Journal: JCI Insight

    doi: 10.1172/jci.insight.125638

    AACOCF3 suppressed phosphate-induced calcification and osteogenic/chondrogenic signaling in HAoSMCs. Representative images showing alizarin red staining ( A , n = 3) and scatter dot plots (arithmetic mean ± SEM) of calcium content ( B , n = 4, μg/mg protein) in HAoSMCs following treatment with control (CTR) or with calcification medium (Calc.), without or with additional treatment with 10 μM AACOCF3 (AAC). The calcified areas are shown as red staining. ( C – G ) Scatter dot plots (arithmetic mean ± SEM) of MSX2 ( C ), CBFA1 ( D ), and ALPL ( E ) relative mRNA expression ( n = 6; arbitrary units [a.u.]) and ALPL activity ( F , n = 4, U/mg protein) in HAoSMCs as well as arachidonic acid (AA) levels ( G , n = 4; ng/mg protein) in the cell culture medium of HAoSMCs following treatment with control or with β-glycerophosphate (Pi), without or with additional treatment with 10 μM AACOCF3. * P
    Figure Legend Snippet: AACOCF3 suppressed phosphate-induced calcification and osteogenic/chondrogenic signaling in HAoSMCs. Representative images showing alizarin red staining ( A , n = 3) and scatter dot plots (arithmetic mean ± SEM) of calcium content ( B , n = 4, μg/mg protein) in HAoSMCs following treatment with control (CTR) or with calcification medium (Calc.), without or with additional treatment with 10 μM AACOCF3 (AAC). The calcified areas are shown as red staining. ( C – G ) Scatter dot plots (arithmetic mean ± SEM) of MSX2 ( C ), CBFA1 ( D ), and ALPL ( E ) relative mRNA expression ( n = 6; arbitrary units [a.u.]) and ALPL activity ( F , n = 4, U/mg protein) in HAoSMCs as well as arachidonic acid (AA) levels ( G , n = 4; ng/mg protein) in the cell culture medium of HAoSMCs following treatment with control or with β-glycerophosphate (Pi), without or with additional treatment with 10 μM AACOCF3. * P

    Techniques Used: Staining, Expressing, Activity Assay, Cell Culture

    Silencing of cPLA2 inhibited phosphate-induced osteogenic/chondrogenic signaling in HAoSMCs. Scatter dot plots (arithmetic mean ± SEM) ( n = 6, arbitrary units [a.u.]) of PLA2G4A ( A ), MSX2 ( B ), CBFA1 ( C ), and ALPL ( D ) relative mRNA expression in HAoSMCs following silencing with negative control siRNA (Neg.si) or cPLA2 siRNA (cPLA2si), without or with additional treatment with β-glycerophosphate (Pi). * P
    Figure Legend Snippet: Silencing of cPLA2 inhibited phosphate-induced osteogenic/chondrogenic signaling in HAoSMCs. Scatter dot plots (arithmetic mean ± SEM) ( n = 6, arbitrary units [a.u.]) of PLA2G4A ( A ), MSX2 ( B ), CBFA1 ( C ), and ALPL ( D ) relative mRNA expression in HAoSMCs following silencing with negative control siRNA (Neg.si) or cPLA2 siRNA (cPLA2si), without or with additional treatment with β-glycerophosphate (Pi). * P

    Techniques Used: Expressing, Negative Control

    17) Product Images from "The Role of PIN1 on Odontogenic and Adipogenic Differentiation in Human Dental Pulp Stem Cells"

    Article Title: The Role of PIN1 on Odontogenic and Adipogenic Differentiation in Human Dental Pulp Stem Cells

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2013.0339

    Time course expression of PIN1 during odontogenic (A) and adipogenic (B) differentiation in HDPSCs. For odontogenic differentiation, cells were cultured in OM containing 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid, and 100 nM dexamethasone for indicated times. For adipogenic differentiation, cells were cultured in adipogenic induction media (high DMEM, 10% FBS, 1 μM dexamethasone, 10 μg/mL insulin, 100 μM indomethacin, and 0.5 mM methyl-isobutylzanthine) for 3 days, and AM (high glucose DMEM, 10% FBS, and 10 μg/mL insulin) for 11 days. These data are representative of three independent experiments. HDPSCs, human dental pulp stem cells; DMEM, Dulbecco's modified Eagle's medium; PIN1, peptidyl-prolyl cis/trans isomerase NIMA-interacting 1; FBS, fetal bovine serum; OM, osteogenic medium; AM, adipogenic induction medium.
    Figure Legend Snippet: Time course expression of PIN1 during odontogenic (A) and adipogenic (B) differentiation in HDPSCs. For odontogenic differentiation, cells were cultured in OM containing 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid, and 100 nM dexamethasone for indicated times. For adipogenic differentiation, cells were cultured in adipogenic induction media (high DMEM, 10% FBS, 1 μM dexamethasone, 10 μg/mL insulin, 100 μM indomethacin, and 0.5 mM methyl-isobutylzanthine) for 3 days, and AM (high glucose DMEM, 10% FBS, and 10 μg/mL insulin) for 11 days. These data are representative of three independent experiments. HDPSCs, human dental pulp stem cells; DMEM, Dulbecco's modified Eagle's medium; PIN1, peptidyl-prolyl cis/trans isomerase NIMA-interacting 1; FBS, fetal bovine serum; OM, osteogenic medium; AM, adipogenic induction medium.

    Techniques Used: Expressing, Cell Culture, Modification

    18) Product Images from "Zinc Inhibits Phosphate-Induced Vascular Calcification through TNFAIP3-Mediated Suppression of NF-κB"

    Article Title: Zinc Inhibits Phosphate-Induced Vascular Calcification through TNFAIP3-Mediated Suppression of NF-κB

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2017050492

    ZnSO 4 inhibits phosphate-induced NF- κ B activation and upregulates TNFAIP3 expression in HAoSMCs. (A) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of normalized phospho-I κ B α )/I κ B α /GAPDH and total I κ B α /GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B) Representative confocal microscopy images showing NF- κ B p65 protein expression and localization in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . Images are representative for three independent experiments. NF- κ B p65 expression: green labeling, nuclei: purple labeling. Scale bar, 25 μ m. (C) Scatterdot plots and arithmetic means±SEM ( n =4; a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (D) Scatterdot plots and arithmetic means±SEM ( n =6; a.u.) of TNFAIP3 relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =5; a.u.) of normalized TNFAIP3/GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . * P
    Figure Legend Snippet: ZnSO 4 inhibits phosphate-induced NF- κ B activation and upregulates TNFAIP3 expression in HAoSMCs. (A) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of normalized phospho-I κ B α )/I κ B α /GAPDH and total I κ B α /GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B) Representative confocal microscopy images showing NF- κ B p65 protein expression and localization in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . Images are representative for three independent experiments. NF- κ B p65 expression: green labeling, nuclei: purple labeling. Scale bar, 25 μ m. (C) Scatterdot plots and arithmetic means±SEM ( n =4; a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (D) Scatterdot plots and arithmetic means±SEM ( n =6; a.u.) of TNFAIP3 relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =5; a.u.) of normalized TNFAIP3/GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . * P

    Techniques Used: Activation Assay, Expressing, Western Blot, Confocal Microscopy, Labeling, Activity Assay, Luciferase, Reporter Assay, Transfection, Construct

    Silencing of TNFAIP3 blunts the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs, silencing for 48 hours with negative control siRNA (Neg.si) or TNFAIP3 siRNA (A20si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B–D) Scatterdot plots and arithmetic means±SEM ( n =8; a.u.) of MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with Neg.si or A20si and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Scatterdot plots and arithmetic means±SEM ( n =6, micrograms per milligram protein) of calcium content in HAoSMCs after silencing for 11 days with Neg.si or A20si and treatment with control or with calcification medium (Calc.) without or with additional treatment with 15 µ M ZnSO 4 . * P
    Figure Legend Snippet: Silencing of TNFAIP3 blunts the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs, silencing for 48 hours with negative control siRNA (Neg.si) or TNFAIP3 siRNA (A20si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B–D) Scatterdot plots and arithmetic means±SEM ( n =8; a.u.) of MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with Neg.si or A20si and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Scatterdot plots and arithmetic means±SEM ( n =6, micrograms per milligram protein) of calcium content in HAoSMCs after silencing for 11 days with Neg.si or A20si and treatment with control or with calcification medium (Calc.) without or with additional treatment with 15 µ M ZnSO 4 . * P

    Techniques Used: Activity Assay, Luciferase, Reporter Assay, Transfection, Construct, Negative Control, Expressing

    Silencing of zinc-sensing receptor ZnR/GPR39 blunts the ZnSO 4 -induced TNFAIP3 expression and the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling in HAoSMCs. Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of TNFAIP3 (A), MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with negative control siRNA (Neg.si) or GPR39 siRNA (GPR39si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . ** P
    Figure Legend Snippet: Silencing of zinc-sensing receptor ZnR/GPR39 blunts the ZnSO 4 -induced TNFAIP3 expression and the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling in HAoSMCs. Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of TNFAIP3 (A), MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with negative control siRNA (Neg.si) or GPR39 siRNA (GPR39si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . ** P

    Techniques Used: Expressing, Negative Control

    ZnSO 4 inhibits phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Representative original images showing Alizarin Red staining in HAoSMCs after treatment for 11 days with control or with calcification medium (Calc.) without or with additional treatment with 1 or 15 µ M ZnSO 4 . Images are representative of four independent experiments. The calcified areas are shown as red staining. (B) Scatterdot plots and arithmetic means±SEM ( n =4, micrograms per milligram protein) of calcium content in HAoSMCs after treatment for 11 days with control or with Calc. without or with additional treatment with 1 or 15 µ M ZnSO 4 . (C) Scatterdot plots and arithmetic means±SEM ( n =4, units per milligram protein) of alkaline phosphatase activity in HAoSMCs after treatment for 7 days with control or with β -glycerophosphate (Pi) without or with additional treatment with 1 or 15 µ M ZnSO 4 . (D–F) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of MSX2 (D), CBFA1 (E), and ALPL (F) relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 1 or 15 µ M ZnSO 4 . * P
    Figure Legend Snippet: ZnSO 4 inhibits phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Representative original images showing Alizarin Red staining in HAoSMCs after treatment for 11 days with control or with calcification medium (Calc.) without or with additional treatment with 1 or 15 µ M ZnSO 4 . Images are representative of four independent experiments. The calcified areas are shown as red staining. (B) Scatterdot plots and arithmetic means±SEM ( n =4, micrograms per milligram protein) of calcium content in HAoSMCs after treatment for 11 days with control or with Calc. without or with additional treatment with 1 or 15 µ M ZnSO 4 . (C) Scatterdot plots and arithmetic means±SEM ( n =4, units per milligram protein) of alkaline phosphatase activity in HAoSMCs after treatment for 7 days with control or with β -glycerophosphate (Pi) without or with additional treatment with 1 or 15 µ M ZnSO 4 . (D–F) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of MSX2 (D), CBFA1 (E), and ALPL (F) relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 1 or 15 µ M ZnSO 4 . * P

    Techniques Used: Staining, Activity Assay, Expressing

    ZnSO 4  inhibits phosphate-induced NF- κ B activation and upregulates  TNFAIP3  expression in HAoSMCs. (A) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of normalized phospho-I κ B α  )/I κ B α /GAPDH and total I κ B α /GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with  β -glycerophosphate (Pi) without or with additional treatment with 15  µ M ZnSO 4 . (B) Representative confocal microscopy images showing NF- κ B p65 protein expression and localization in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15  µ M ZnSO 4 . Images are representative for three independent experiments. NF- κ B p65 expression: green labeling, nuclei: purple labeling. Scale bar, 25  μ m. (C) Scatterdot plots and arithmetic means±SEM ( n =4; a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla  constructs and treatment for 24 hours with control or with Pi without or with additional treatment with 15  µ M ZnSO 4 . (D) Scatterdot plots and arithmetic means±SEM ( n =6; a.u.) of  TNFAIP3  relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15  µ M ZnSO 4 . (E) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =5; a.u.) of normalized TNFAIP3/GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15  µ M ZnSO 4 . * P
    Figure Legend Snippet: ZnSO 4 inhibits phosphate-induced NF- κ B activation and upregulates TNFAIP3 expression in HAoSMCs. (A) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of normalized phospho-I κ B α )/I κ B α /GAPDH and total I κ B α /GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B) Representative confocal microscopy images showing NF- κ B p65 protein expression and localization in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . Images are representative for three independent experiments. NF- κ B p65 expression: green labeling, nuclei: purple labeling. Scale bar, 25 μ m. (C) Scatterdot plots and arithmetic means±SEM ( n =4; a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (D) Scatterdot plots and arithmetic means±SEM ( n =6; a.u.) of TNFAIP3 relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =5; a.u.) of normalized TNFAIP3/GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . * P

    Techniques Used: Activation Assay, Expressing, Western Blot, Confocal Microscopy, Labeling, Activity Assay, Luciferase, Reporter Assay, Transfection, Construct

    Silencing of TNFAIP3 blunts the protective effects of ZnSO 4  on phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla  constructs, silencing for 48 hours with negative control siRNA (Neg.si) or TNFAIP3 siRNA (A20si) and treatment for 24 hours with control or with  β -glycerophosphate (Pi) without or with additional treatment with 15  µ M ZnSO 4 . (B–D) Scatterdot plots and arithmetic means±SEM ( n =8; a.u.) of  MSX2  (B),  CBFA1  (C), and  ALPL  (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with Neg.si or A20si and treatment for 24 hours with control or with Pi without or with additional treatment with 15  µ M ZnSO 4 . (E) Scatterdot plots and arithmetic means±SEM ( n =6, micrograms per milligram protein) of calcium content in HAoSMCs after silencing for 11 days with Neg.si or A20si and treatment with control or with calcification medium (Calc.) without or with additional treatment with 15  µ M ZnSO 4 . * P
    Figure Legend Snippet: Silencing of TNFAIP3 blunts the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs, silencing for 48 hours with negative control siRNA (Neg.si) or TNFAIP3 siRNA (A20si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B–D) Scatterdot plots and arithmetic means±SEM ( n =8; a.u.) of MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with Neg.si or A20si and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Scatterdot plots and arithmetic means±SEM ( n =6, micrograms per milligram protein) of calcium content in HAoSMCs after silencing for 11 days with Neg.si or A20si and treatment with control or with calcification medium (Calc.) without or with additional treatment with 15 µ M ZnSO 4 . * P

    Techniques Used: Activity Assay, Luciferase, Reporter Assay, Transfection, Construct, Negative Control, Expressing

    Silencing of zinc-sensing receptor ZnR/GPR39 blunts the ZnSO 4 -induced  TNFAIP3  expression and the protective effects of ZnSO 4  on phosphate-induced osteoinductive signaling in HAoSMCs. Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of  TNFAIP3  (A),  MSX2  (B),  CBFA1  (C), and  ALPL  (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with negative control siRNA (Neg.si) or GPR39 siRNA (GPR39si) and treatment for 24 hours with control or with  β -glycerophosphate (Pi) without or with additional treatment with 15  µ M ZnSO 4 . ** P
    Figure Legend Snippet: Silencing of zinc-sensing receptor ZnR/GPR39 blunts the ZnSO 4 -induced TNFAIP3 expression and the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling in HAoSMCs. Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of TNFAIP3 (A), MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with negative control siRNA (Neg.si) or GPR39 siRNA (GPR39si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . ** P

    Techniques Used: Expressing, Negative Control

    ZnSO 4  inhibits phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Representative original images showing Alizarin Red staining in HAoSMCs after treatment for 11 days with control or with calcification medium (Calc.) without or with additional treatment with 1 or 15  µ M ZnSO 4 . Images are representative of four independent experiments. The calcified areas are shown as red staining. (B) Scatterdot plots and arithmetic means±SEM ( n =4, micrograms per milligram protein) of calcium content in HAoSMCs after treatment for 11 days with control or with Calc. without or with additional treatment with 1 or 15  µ M ZnSO 4 . (C) Scatterdot plots and arithmetic means±SEM ( n =4, units per milligram protein) of alkaline phosphatase activity in HAoSMCs after treatment for 7 days with control or with  β -glycerophosphate (Pi) without or with additional treatment with 1 or 15  µ M ZnSO 4 . (D–F) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of  MSX2  (D),  CBFA1  (E), and  ALPL  (F) relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 1 or 15  µ M ZnSO 4 . * P
    Figure Legend Snippet: ZnSO 4 inhibits phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Representative original images showing Alizarin Red staining in HAoSMCs after treatment for 11 days with control or with calcification medium (Calc.) without or with additional treatment with 1 or 15 µ M ZnSO 4 . Images are representative of four independent experiments. The calcified areas are shown as red staining. (B) Scatterdot plots and arithmetic means±SEM ( n =4, micrograms per milligram protein) of calcium content in HAoSMCs after treatment for 11 days with control or with Calc. without or with additional treatment with 1 or 15 µ M ZnSO 4 . (C) Scatterdot plots and arithmetic means±SEM ( n =4, units per milligram protein) of alkaline phosphatase activity in HAoSMCs after treatment for 7 days with control or with β -glycerophosphate (Pi) without or with additional treatment with 1 or 15 µ M ZnSO 4 . (D–F) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of MSX2 (D), CBFA1 (E), and ALPL (F) relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 1 or 15 µ M ZnSO 4 . * P

    Techniques Used: Staining, Activity Assay, Expressing

    19) Product Images from "Impaired bone development and increased mesenchymal progenitor cells in calvaria of RB1−/− mice"

    Article Title: Impaired bone development and increased mesenchymal progenitor cells in calvaria of RB1−/− mice

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0805925105

    Proliferative properties and increased mineral deposition in cultured primary calvarial osteoblasts ( A ). Immunoblot analysis of extracts of cultured osteoblasts shows the absence of pRb in col3.6;RB1 lox/lox mice. ( B ) Osteoblasts were isolated from the calvaria of E18.5 animals and differentiated in α-MEM media supplemented with ascorbic acid and β-glycerophosphate. Total cell numbers were determined at 1, 7, 14, 21, and 28 days after plating at an initial density of 500 cells/cm 2 . *, P
    Figure Legend Snippet: Proliferative properties and increased mineral deposition in cultured primary calvarial osteoblasts ( A ). Immunoblot analysis of extracts of cultured osteoblasts shows the absence of pRb in col3.6;RB1 lox/lox mice. ( B ) Osteoblasts were isolated from the calvaria of E18.5 animals and differentiated in α-MEM media supplemented with ascorbic acid and β-glycerophosphate. Total cell numbers were determined at 1, 7, 14, 21, and 28 days after plating at an initial density of 500 cells/cm 2 . *, P

    Techniques Used: Cell Culture, Mouse Assay, Isolation

    20) Product Images from "Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2β1 Signaling"

    Article Title: Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2β1 Signaling

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2015-0412

    Adherent and spheroidal mesenchymal stem cells (MSCs) were encapsulated within collagen I hydrogels and cultured in three medium regimens. After 12 days of culture in growth or osteogenic media, cells were trypsinized and formed into spheroids or replated in adherent monolayer. After 2 days of spheroidal or adherent culture in the same media, cells were encapsulated within collagen gels. (A): MSCs cultured in growth media for the full duration (GM) served as the negative control. (B): Osteogenic media were replaced with growth media for an additional 5 days (+5d) (OM/GM). (C): As a positive control, osteogenically induced MSCs remained in OM. Osteogenic markers were assessed at +1d and +5d, where 0d marks the day cells were entrapped in hydrogels and osteogenic media were switched to growth media (OM/GM). Abbreviations: ASP, ascorbate-2-phosphate; BGP, β-glycerophosphate; d, day; Dex, dexamethasone; GM, growth media; OM, osteogenic media.
    Figure Legend Snippet: Adherent and spheroidal mesenchymal stem cells (MSCs) were encapsulated within collagen I hydrogels and cultured in three medium regimens. After 12 days of culture in growth or osteogenic media, cells were trypsinized and formed into spheroids or replated in adherent monolayer. After 2 days of spheroidal or adherent culture in the same media, cells were encapsulated within collagen gels. (A): MSCs cultured in growth media for the full duration (GM) served as the negative control. (B): Osteogenic media were replaced with growth media for an additional 5 days (+5d) (OM/GM). (C): As a positive control, osteogenically induced MSCs remained in OM. Osteogenic markers were assessed at +1d and +5d, where 0d marks the day cells were entrapped in hydrogels and osteogenic media were switched to growth media (OM/GM). Abbreviations: ASP, ascorbate-2-phosphate; BGP, β-glycerophosphate; d, day; Dex, dexamethasone; GM, growth media; OM, osteogenic media.

    Techniques Used: Cell Culture, Negative Control, Positive Control

    21) Product Images from "Degradation, Bioactivity, and Osteogenic Potential of Composites Made of PLGA and Two Different Sol-Gel Bioactive Glasses"

    Article Title: Degradation, Bioactivity, and Osteogenic Potential of Composites Made of PLGA and Two Different Sol-Gel Bioactive Glasses

    Journal: Annals of Biomedical Engineering

    doi: 10.1007/s10439-011-0307-4

    Phalloidin/DAPI staining (a) of MG-63 cells cultured in 3D PLGA, PLGA/S2-21, and PLGA/A2-21 scaffolds and stimulated with ascorbate, dexamethasone, and β-glycerophosphate for 2 weeks. Cells were also analyzed for cell number (b), collagen (c), and calcium (d) levels after 2-week culture. Results for b–d are expressed as mean ± S.E. Statistically significant differences ( p
    Figure Legend Snippet: Phalloidin/DAPI staining (a) of MG-63 cells cultured in 3D PLGA, PLGA/S2-21, and PLGA/A2-21 scaffolds and stimulated with ascorbate, dexamethasone, and β-glycerophosphate for 2 weeks. Cells were also analyzed for cell number (b), collagen (c), and calcium (d) levels after 2-week culture. Results for b–d are expressed as mean ± S.E. Statistically significant differences ( p

    Techniques Used: Staining, Cell Culture

    22) Product Images from "Exogenous Stimulation of Human Intervertebral Disc Cells in 3-Dimensional Alginate Bead Culture With BMP2 and L51P: Cytocompatibility and Effects on Cell Phenotype"

    Article Title: Exogenous Stimulation of Human Intervertebral Disc Cells in 3-Dimensional Alginate Bead Culture With BMP2 and L51P: Cytocompatibility and Effects on Cell Phenotype

    Journal: Neurospine

    doi: 10.14245/ns.2040002.001

    Experimental design of human intervertebral disc cells, i.e., nucleus pulposus (NPCs), annulus fibrosus (AFCs), and cartilaginous endplate cells (CEPCs) (encapsulated in 1.2% alginate) stimulation with 100-ng/mL bone morphogenetic protein 2 (BMP2) and/or L51P in osteogenic or basal medium for 21 days. Not shown are the 3 controls, basal and osteogenic medium only stimulation and β-glycerophosphate control (α-minimal essential medium supplemented with 10% foetal bovine serum, penicillin/streptomycin, and 5 mM β-glycerophosphate).
    Figure Legend Snippet: Experimental design of human intervertebral disc cells, i.e., nucleus pulposus (NPCs), annulus fibrosus (AFCs), and cartilaginous endplate cells (CEPCs) (encapsulated in 1.2% alginate) stimulation with 100-ng/mL bone morphogenetic protein 2 (BMP2) and/or L51P in osteogenic or basal medium for 21 days. Not shown are the 3 controls, basal and osteogenic medium only stimulation and β-glycerophosphate control (α-minimal essential medium supplemented with 10% foetal bovine serum, penicillin/streptomycin, and 5 mM β-glycerophosphate).

    Techniques Used:

    Alcian blue staining for sulfated glycosaminoglycan visualization. Nucleus pulposus (NPC), annulus fibrosus (AFC), and cartilaginous endplate cell (CEPC) beads stimulated basal medium (BM) supplemented with 100 ng/mL of bone morphogenetic protein 2 (BMP2) and/or L51P. β-GP, β-glycerophosphate.
    Figure Legend Snippet: Alcian blue staining for sulfated glycosaminoglycan visualization. Nucleus pulposus (NPC), annulus fibrosus (AFC), and cartilaginous endplate cell (CEPC) beads stimulated basal medium (BM) supplemented with 100 ng/mL of bone morphogenetic protein 2 (BMP2) and/or L51P. β-GP, β-glycerophosphate.

    Techniques Used: Staining

    23) Product Images from "Calcite incorporated in silica/collagen xerogels mediates calcium release and enhances osteoblast proliferation and differentiation"

    Article Title: Calcite incorporated in silica/collagen xerogels mediates calcium release and enhances osteoblast proliferation and differentiation

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-56023-8

    Proliferation ( a , b , e , f ) and osteogenic differentiation ( c , d , g , h ) of hMSC cultured in presence (indirect cell culture, a – d ) and on the surface (direct cell culture, e – h ) of B30, B30H20 and B30CK20. Attachment of hMSC on the surface of B30 ( i ), B30H20 ( j ) and B30CK20 ( k ), 24 h after seeding. Cells were seeded at 1.7 × 10 4 per 24-well and 2 × 10 4 per xerogel, respectively and were treated with 50 µM ascorbate on the first day of adherence (Os−; a , c , e , g ). For osteogenic differentiation (Os+; b , d , f , h ), hMSC were treated with 10 nM dexamethasone, 5 mM β-glycerophosphate and 50 µM ascorbate by day 3. Number of cells and ALP activity were measured from cell lysates by biochemical assays. * p
    Figure Legend Snippet: Proliferation ( a , b , e , f ) and osteogenic differentiation ( c , d , g , h ) of hMSC cultured in presence (indirect cell culture, a – d ) and on the surface (direct cell culture, e – h ) of B30, B30H20 and B30CK20. Attachment of hMSC on the surface of B30 ( i ), B30H20 ( j ) and B30CK20 ( k ), 24 h after seeding. Cells were seeded at 1.7 × 10 4 per 24-well and 2 × 10 4 per xerogel, respectively and were treated with 50 µM ascorbate on the first day of adherence (Os−; a , c , e , g ). For osteogenic differentiation (Os+; b , d , f , h ), hMSC were treated with 10 nM dexamethasone, 5 mM β-glycerophosphate and 50 µM ascorbate by day 3. Number of cells and ALP activity were measured from cell lysates by biochemical assays. * p

    Techniques Used: Cell Culture, ALP Assay, Activity Assay

    24) Product Images from "Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells "

    Article Title: Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells

    Journal: The Journal of Cell Biology

    doi:

    Titration conditions for recombinant CAF-1 to support nucleosome assembly during UV-induced synthesis and competence of various phosphorylated forms in the assay. ( A ) Plasmid DNA, either treated with UV-C at 500 J/m 2 (+) or not treated (−) were incubated in a cytosolic extract in the presence of [α- 32 P] dCTP. Complementation in the supercoiling assay was achieved with increasing amounts of recombinant CAF-1 ( solid triangle ), 0 ng (lanes 2 , 3 , and 4 ), 5 ng (lane 5 ), 10 ng (lane 6 ), 20 ng (lane 7 ), or 40 ng (lane 8 ). Deproteinized DNA was purified and analyzed by agarose gel electrophoresis. The incorporation of radiolabel due to the UV-dependent DNA synthesis was visualized by autoradiography ( Labeled DNA ) and the total population of DNA molecules by ethidium bromide staining of the gel ( Total DNA ). Plasmids were processed as indicated in Materials and Methods. The positions of the supercoiled (form I ), nicked (form Ir ), and closed circular (form II ) forms of plasmid DNA are indicated. Control supercoiled DNA was run in parallel (lane 1 ). ( B ) Recombinant CAF-1 was treated with Lambda phosphatase alone (lane 1 ), in the presence of β-glycerophosphate (lane 2 ) or mock treated (lanes 3 and 5 ), mitotic extract ( M ) and nuclear interphasic extract ( I ) were prepared as described (see Materials and Methods). All samples were processed for Western blot analysis and detection with the polyclonal anti–p60 antibody ( p60 ). Bracket , various phosphorylation forms. ( C ) The three sources of recombinant CAF-1 ( r.CAF-1 ): treated with Lambda phosphatase alone (lane 4 ), in the presence of β-glycerophosphate (lane 5 ), or mock treated (lane 3 ) (shown in B , left ) were used in the assay as described above. The amount added per reaction was 10 ng (defined as the critical amount in A ). The presence and amount of the phosphorylated p60 subunit of CAF-1 (exogenous and endogenous) at the end of each reaction was assessed by Western blotting using the polyclonal anti-p60 antibody ( p60 ). Asterisk , slowest migrating phosphorylated form of p60.
    Figure Legend Snippet: Titration conditions for recombinant CAF-1 to support nucleosome assembly during UV-induced synthesis and competence of various phosphorylated forms in the assay. ( A ) Plasmid DNA, either treated with UV-C at 500 J/m 2 (+) or not treated (−) were incubated in a cytosolic extract in the presence of [α- 32 P] dCTP. Complementation in the supercoiling assay was achieved with increasing amounts of recombinant CAF-1 ( solid triangle ), 0 ng (lanes 2 , 3 , and 4 ), 5 ng (lane 5 ), 10 ng (lane 6 ), 20 ng (lane 7 ), or 40 ng (lane 8 ). Deproteinized DNA was purified and analyzed by agarose gel electrophoresis. The incorporation of radiolabel due to the UV-dependent DNA synthesis was visualized by autoradiography ( Labeled DNA ) and the total population of DNA molecules by ethidium bromide staining of the gel ( Total DNA ). Plasmids were processed as indicated in Materials and Methods. The positions of the supercoiled (form I ), nicked (form Ir ), and closed circular (form II ) forms of plasmid DNA are indicated. Control supercoiled DNA was run in parallel (lane 1 ). ( B ) Recombinant CAF-1 was treated with Lambda phosphatase alone (lane 1 ), in the presence of β-glycerophosphate (lane 2 ) or mock treated (lanes 3 and 5 ), mitotic extract ( M ) and nuclear interphasic extract ( I ) were prepared as described (see Materials and Methods). All samples were processed for Western blot analysis and detection with the polyclonal anti–p60 antibody ( p60 ). Bracket , various phosphorylation forms. ( C ) The three sources of recombinant CAF-1 ( r.CAF-1 ): treated with Lambda phosphatase alone (lane 4 ), in the presence of β-glycerophosphate (lane 5 ), or mock treated (lane 3 ) (shown in B , left ) were used in the assay as described above. The amount added per reaction was 10 ng (defined as the critical amount in A ). The presence and amount of the phosphorylated p60 subunit of CAF-1 (exogenous and endogenous) at the end of each reaction was assessed by Western blotting using the polyclonal anti-p60 antibody ( p60 ). Asterisk , slowest migrating phosphorylated form of p60.

    Techniques Used: Titration, Recombinant, Plasmid Preparation, Incubation, Purification, Agarose Gel Electrophoresis, DNA Synthesis, Autoradiography, Labeling, Staining, Western Blot

    25) Product Images from "TGF-? Inhibition Restores Terminal Osteoblast Differentiation to Suppress Myeloma Growth"

    Article Title: TGF-? Inhibition Restores Terminal Osteoblast Differentiation to Suppress Myeloma Growth

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009870

    TGF-β suppresses and TGF-β inhibitors enhance OB differentiation. MC3T3-E1 cells ( A ) and primary bone marrow stromal cells ( B ) were cultured for 14 and 28 days, respectively, in 24-well culture plates in α-MEM containing 10% FBS supplemented with β-glycerophosphate and ascorbic acid (osteogenic medium). rhBMP-2, SB431542, Ki26894 and rhTGF-β were added at 50 ng/mL, 3 µM, 10 µM and 5 ng/mL to the indicated wells, respectively. After culturing, mineralized nodules were visualized by von Kossa staining. C. MC3T3-E1 cells were cultured for 21 days in osteogenic media in the absence of BMP-2. SB431542 was added at 3 µM to the indicated wells. Mineralized nodules were visualized by von Kossa staining. The data with mineralized nodule formation were quantified by densitometric analyses.
    Figure Legend Snippet: TGF-β suppresses and TGF-β inhibitors enhance OB differentiation. MC3T3-E1 cells ( A ) and primary bone marrow stromal cells ( B ) were cultured for 14 and 28 days, respectively, in 24-well culture plates in α-MEM containing 10% FBS supplemented with β-glycerophosphate and ascorbic acid (osteogenic medium). rhBMP-2, SB431542, Ki26894 and rhTGF-β were added at 50 ng/mL, 3 µM, 10 µM and 5 ng/mL to the indicated wells, respectively. After culturing, mineralized nodules were visualized by von Kossa staining. C. MC3T3-E1 cells were cultured for 21 days in osteogenic media in the absence of BMP-2. SB431542 was added at 3 µM to the indicated wells. Mineralized nodules were visualized by von Kossa staining. The data with mineralized nodule formation were quantified by densitometric analyses.

    Techniques Used: Cell Culture, Staining

    Terminally differentiated OBs suppress MM cell growth. A. MC3T3-E1 cells and primary bone marrow stromal cells isolated from a patient with MM were cultured in 24-well culture plates in α-MEM containing 10% FBS with or without β-glycerophosphate, ascorbic acid and 50 ng/mL rhBMP-2 as described in “ Materials and methods ”. After forming mineralized nodules in the osteogenic cultures with rhBMP-2, the cells were washed to remove rhBMP-2. MM cell lines, 5TGM1, RPMI8226, U266 and KMS-12 at 5×10 4 /mL, were cultured alone as a control or cocultured in quadruplicate with thus treated MC3T3-E1 cells or primary bone marrow stromal cells with or without inducing terminal OB differentiation. After culturing for 3 days, MM cells were harvested, and viable MM cell numbers were counted. Percent changes from the control are shown. Results are expressed as means +/− SD. *,
    Figure Legend Snippet: Terminally differentiated OBs suppress MM cell growth. A. MC3T3-E1 cells and primary bone marrow stromal cells isolated from a patient with MM were cultured in 24-well culture plates in α-MEM containing 10% FBS with or without β-glycerophosphate, ascorbic acid and 50 ng/mL rhBMP-2 as described in “ Materials and methods ”. After forming mineralized nodules in the osteogenic cultures with rhBMP-2, the cells were washed to remove rhBMP-2. MM cell lines, 5TGM1, RPMI8226, U266 and KMS-12 at 5×10 4 /mL, were cultured alone as a control or cocultured in quadruplicate with thus treated MC3T3-E1 cells or primary bone marrow stromal cells with or without inducing terminal OB differentiation. After culturing for 3 days, MM cells were harvested, and viable MM cell numbers were counted. Percent changes from the control are shown. Results are expressed as means +/− SD. *,

    Techniques Used: Isolation, Cell Culture

    26) Product Images from "Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes"

    Article Title: Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200204060

    von Kossa staining of confluent LL, II-RIA, and II-RIB cells and microscopic visualization of the differentiated osteoblast phenotype. (A) von Kossa staining of LL, II-RIA, and II-RIB cells, cultured in the presence of β-glycerophosphate and ascorbic acid for 25 d, in the absence or presence of 10 −6 M retinoic acid. (B) Microscopic visualization of LL or II-RIB cells cultured for 25 d in the presence of β-glycerophosphate and ascorbic acid, and 10 −6 M retinoic acid. RA, retinoic acid.
    Figure Legend Snippet: von Kossa staining of confluent LL, II-RIA, and II-RIB cells and microscopic visualization of the differentiated osteoblast phenotype. (A) von Kossa staining of LL, II-RIA, and II-RIB cells, cultured in the presence of β-glycerophosphate and ascorbic acid for 25 d, in the absence or presence of 10 −6 M retinoic acid. (B) Microscopic visualization of LL or II-RIB cells cultured for 25 d in the presence of β-glycerophosphate and ascorbic acid, and 10 −6 M retinoic acid. RA, retinoic acid.

    Techniques Used: Staining, Cell Culture

    27) Product Images from "Ecto-alkaline phosphatase in NG108-15 cells : a key enzyme mediating P1 antagonist-sensitive ATP response"

    Article Title: Ecto-alkaline phosphatase in NG108-15 cells : a key enzyme mediating P1 antagonist-sensitive ATP response

    Journal: British Journal of Pharmacology

    doi: 10.1038/sj.bjp.0703750

    Hydrolysis of AMP, 3′-AMP (A3P), ribose-5-phosphate (R-5-P) and β-glycerophosphate (β-GP) in NG108-15 cells. Aliquots of suspended cells were incubated with different concentrations of AMP, A3P, R-5-P and β-GP for 10 min at 37°C, and free orthophosphate levels in the medium measured. Data are mean of duplicate determinations and are typical of three independent experiments.
    Figure Legend Snippet: Hydrolysis of AMP, 3′-AMP (A3P), ribose-5-phosphate (R-5-P) and β-glycerophosphate (β-GP) in NG108-15 cells. Aliquots of suspended cells were incubated with different concentrations of AMP, A3P, R-5-P and β-GP for 10 min at 37°C, and free orthophosphate levels in the medium measured. Data are mean of duplicate determinations and are typical of three independent experiments.

    Techniques Used: Incubation

    28) Product Images from "SGK1 induces vascular smooth muscle cell calcification through NF- κB signaling"

    Article Title: SGK1 induces vascular smooth muscle cell calcification through NF- κB signaling

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI96477

    SGK1 inhibition ameliorates phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary HAoSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 7 per group; AU) of SGK1 relative mRNA expression in HAoSMCs following treatment with control or β-glycerophosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). ( B ) Representative confocal microscopy images ( n = 3 per group) showing NF-κB p65 protein expression and localization in HAoSMCs following treatment with control or β-glycerophosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). Green labeling, NF-κB p65 expression; magenta labeling, nuclei. Scale bars: 20 μm. ( C ) Representative original images ( n = 4 per group) showing alizarin red staining in HAoSMCs following treatment with control or with calcification medium without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). Calcified areas are shown as red staining. ( D – H ) Scatter dot plots and arithmetic means ± SEM of calcium content ( D , n = 6 per group, μg/mg protein), alkaline phosphatase activity ( E , n = 4 per group, U/mg protein), and MSX2 ( F ), CBFA1 ( G ), and ALPL ( H ) relative mRNA expression ( n = 7 per group; AU) in HAoSMCs following treatment with control or phosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). * P
    Figure Legend Snippet: SGK1 inhibition ameliorates phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary HAoSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 7 per group; AU) of SGK1 relative mRNA expression in HAoSMCs following treatment with control or β-glycerophosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). ( B ) Representative confocal microscopy images ( n = 3 per group) showing NF-κB p65 protein expression and localization in HAoSMCs following treatment with control or β-glycerophosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). Green labeling, NF-κB p65 expression; magenta labeling, nuclei. Scale bars: 20 μm. ( C ) Representative original images ( n = 4 per group) showing alizarin red staining in HAoSMCs following treatment with control or with calcification medium without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). Calcified areas are shown as red staining. ( D – H ) Scatter dot plots and arithmetic means ± SEM of calcium content ( D , n = 6 per group, μg/mg protein), alkaline phosphatase activity ( E , n = 4 per group, U/mg protein), and MSX2 ( F ), CBFA1 ( G ), and ALPL ( H ) relative mRNA expression ( n = 7 per group; AU) in HAoSMCs following treatment with control or phosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). * P

    Techniques Used: Inhibition, Expressing, Confocal Microscopy, Labeling, Staining, Activity Assay

    Sgk1 deficiency reduces phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary MAoSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 6 per group; AU) of Sgk1 relative mRNA expression in primary MAoSMCs isolated from WT mice (sgk1 +/+ ) and treated with control or β-glycerophosphate (Pi). ( B ) Representative confocal microscopy images ( n = 3 per group) showing Sgk1 and NF-κB p65 protein expression and scatter dot plots and arithmetic means ± SEM ( n = 3 per group; AU) of normalized Sgk1 fluorescence intensity in sgk1 –/– or sgk1 +/+ MAoSMCs treated with control or Pi. Green labeling, Sgk1 or NF-κB p65 expression; magenta labeling, nuclei. Scale bars: 20 μm. ** P
    Figure Legend Snippet: Sgk1 deficiency reduces phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary MAoSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 6 per group; AU) of Sgk1 relative mRNA expression in primary MAoSMCs isolated from WT mice (sgk1 +/+ ) and treated with control or β-glycerophosphate (Pi). ( B ) Representative confocal microscopy images ( n = 3 per group) showing Sgk1 and NF-κB p65 protein expression and scatter dot plots and arithmetic means ± SEM ( n = 3 per group; AU) of normalized Sgk1 fluorescence intensity in sgk1 –/– or sgk1 +/+ MAoSMCs treated with control or Pi. Green labeling, Sgk1 or NF-κB p65 expression; magenta labeling, nuclei. Scale bars: 20 μm. ** P

    Techniques Used: Expressing, Isolation, Mouse Assay, Confocal Microscopy, Fluorescence, Labeling

    SGK1 expression in VSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 6 per group; arbitrary units [AU]) of SGK1 relative mRNA expression in primary HAoSMCs following treatment with control (CTR) or aldosterone (Aldo), dexamethasone (Dex), β-glycerophosphate (Pi), glucose, recombinant human TGF-β1 protein, or recombinant human BMP-2 protein. ( B ) Representative original Western blots and scatter dot plots and arithmetic means ± SEM ( n = 4 per group; AU) of normalized SGK1/GAPDH protein ratio in HAoSMCs following treatment with triggers of osteo-chondrogenic transdifferentiation. * P
    Figure Legend Snippet: SGK1 expression in VSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM ( n = 6 per group; arbitrary units [AU]) of SGK1 relative mRNA expression in primary HAoSMCs following treatment with control (CTR) or aldosterone (Aldo), dexamethasone (Dex), β-glycerophosphate (Pi), glucose, recombinant human TGF-β1 protein, or recombinant human BMP-2 protein. ( B ) Representative original Western blots and scatter dot plots and arithmetic means ± SEM ( n = 4 per group; AU) of normalized SGK1/GAPDH protein ratio in HAoSMCs following treatment with triggers of osteo-chondrogenic transdifferentiation. * P

    Techniques Used: Expressing, Recombinant, Western Blot

    29) Product Images from "Type I collagen deposition via osteoinduction ameliorates YAP/TAZ activity in 3D floating culture clumps of mesenchymal stem cell/extracellular matrix complexes"

    Article Title: Type I collagen deposition via osteoinduction ameliorates YAP/TAZ activity in 3D floating culture clumps of mesenchymal stem cell/extracellular matrix complexes

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-1085-9

    The effects of OIM components on COL1 production, F-actin integrity, and YAP/TAZ activity in C-MSCs. a – c C-MSCs were cultured with GM (none) in the presence of β-glycerophosphate (Gly), ascorbic acid (AA), and/or dexamethasone (Dex) (Gly+AA, Gly+Dex, AA+Dex, and Gly+AA+Dex (equivalent to OIM)) for 5 days. a Confocal immunofluorescence images of COL1 (green) and nuclei (blue) in C-MSCs. Bar = 100 μm. b Confocal immunofluorescence images of YAP/TAZ (green), F-actin (red), and nuclei (blue) in C-MSCs. Bar = 20 μm. c The graph summarizes the distribution of YAP/TAZ patterns. The patterns were classified as mainly nuclear ( N > C ), diffuse ( N = C ), and mainly cytoplasmic or undetectable ( N
    Figure Legend Snippet: The effects of OIM components on COL1 production, F-actin integrity, and YAP/TAZ activity in C-MSCs. a – c C-MSCs were cultured with GM (none) in the presence of β-glycerophosphate (Gly), ascorbic acid (AA), and/or dexamethasone (Dex) (Gly+AA, Gly+Dex, AA+Dex, and Gly+AA+Dex (equivalent to OIM)) for 5 days. a Confocal immunofluorescence images of COL1 (green) and nuclei (blue) in C-MSCs. Bar = 100 μm. b Confocal immunofluorescence images of YAP/TAZ (green), F-actin (red), and nuclei (blue) in C-MSCs. Bar = 20 μm. c The graph summarizes the distribution of YAP/TAZ patterns. The patterns were classified as mainly nuclear ( N > C ), diffuse ( N = C ), and mainly cytoplasmic or undetectable ( N

    Techniques Used: Activity Assay, Cell Culture, Immunofluorescence

    30) Product Images from "Age-related Marrow Adipogenesis Is Linked to Increased Expression of RANKL *"

    Article Title: Age-related Marrow Adipogenesis Is Linked to Increased Expression of RANKL *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.547919

    Increased osteoclastogenesis in osteogenic bone marrow cultures of aged mice. A and B , bone marrow cells from 4-month ( 4M )-, 1-year ( 1Y )-, and 2-year ( 2Y )-old mice were cultured under osteogenic conditions with β-glycerophosphate and ascorbic
    Figure Legend Snippet: Increased osteoclastogenesis in osteogenic bone marrow cultures of aged mice. A and B , bone marrow cells from 4-month ( 4M )-, 1-year ( 1Y )-, and 2-year ( 2Y )-old mice were cultured under osteogenic conditions with β-glycerophosphate and ascorbic

    Techniques Used: Mouse Assay, Cell Culture

    31) Product Images from "Zinc Inhibits Phosphate-Induced Vascular Calcification through TNFAIP3-Mediated Suppression of NF-κB"

    Article Title: Zinc Inhibits Phosphate-Induced Vascular Calcification through TNFAIP3-Mediated Suppression of NF-κB

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2017050492

    ZnSO 4 inhibits phosphate-induced NF- κ B activation and upregulates TNFAIP3 expression in HAoSMCs. (A) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of normalized phospho-I κ B α )/I κ B α /GAPDH and total I κ B α /GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B) Representative confocal microscopy images showing NF- κ B p65 protein expression and localization in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . Images are representative for three independent experiments. NF- κ B p65 expression: green labeling, nuclei: purple labeling. Scale bar, 25 μ m. (C) Scatterdot plots and arithmetic means±SEM ( n =4; a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (D) Scatterdot plots and arithmetic means±SEM ( n =6; a.u.) of TNFAIP3 relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =5; a.u.) of normalized TNFAIP3/GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . * P
    Figure Legend Snippet: ZnSO 4 inhibits phosphate-induced NF- κ B activation and upregulates TNFAIP3 expression in HAoSMCs. (A) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of normalized phospho-I κ B α )/I κ B α /GAPDH and total I κ B α /GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B) Representative confocal microscopy images showing NF- κ B p65 protein expression and localization in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . Images are representative for three independent experiments. NF- κ B p65 expression: green labeling, nuclei: purple labeling. Scale bar, 25 μ m. (C) Scatterdot plots and arithmetic means±SEM ( n =4; a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (D) Scatterdot plots and arithmetic means±SEM ( n =6; a.u.) of TNFAIP3 relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =5; a.u.) of normalized TNFAIP3/GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . * P

    Techniques Used: Activation Assay, Expressing, Western Blot, Confocal Microscopy, Labeling, Activity Assay, Luciferase, Reporter Assay, Transfection, Construct

    Silencing of TNFAIP3 blunts the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs, silencing for 48 hours with negative control siRNA (Neg.si) or TNFAIP3 siRNA (A20si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B–D) Scatterdot plots and arithmetic means±SEM ( n =8; a.u.) of MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with Neg.si or A20si and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Scatterdot plots and arithmetic means±SEM ( n =6, micrograms per milligram protein) of calcium content in HAoSMCs after silencing for 11 days with Neg.si or A20si and treatment with control or with calcification medium (Calc.) without or with additional treatment with 15 µ M ZnSO 4 . * P
    Figure Legend Snippet: Silencing of TNFAIP3 blunts the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs, silencing for 48 hours with negative control siRNA (Neg.si) or TNFAIP3 siRNA (A20si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B–D) Scatterdot plots and arithmetic means±SEM ( n =8; a.u.) of MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with Neg.si or A20si and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Scatterdot plots and arithmetic means±SEM ( n =6, micrograms per milligram protein) of calcium content in HAoSMCs after silencing for 11 days with Neg.si or A20si and treatment with control or with calcification medium (Calc.) without or with additional treatment with 15 µ M ZnSO 4 . * P

    Techniques Used: Activity Assay, Luciferase, Reporter Assay, Transfection, Construct, Negative Control, Expressing

    Silencing of zinc-sensing receptor ZnR/GPR39 blunts the ZnSO 4 -induced TNFAIP3 expression and the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling in HAoSMCs. Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of TNFAIP3 (A), MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with negative control siRNA (Neg.si) or GPR39 siRNA (GPR39si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . ** P
    Figure Legend Snippet: Silencing of zinc-sensing receptor ZnR/GPR39 blunts the ZnSO 4 -induced TNFAIP3 expression and the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling in HAoSMCs. Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of TNFAIP3 (A), MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with negative control siRNA (Neg.si) or GPR39 siRNA (GPR39si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . ** P

    Techniques Used: Expressing, Negative Control

    ZnSO 4 inhibits phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Representative original images showing Alizarin Red staining in HAoSMCs after treatment for 11 days with control or with calcification medium (Calc.) without or with additional treatment with 1 or 15 µ M ZnSO 4 . Images are representative of four independent experiments. The calcified areas are shown as red staining. (B) Scatterdot plots and arithmetic means±SEM ( n =4, micrograms per milligram protein) of calcium content in HAoSMCs after treatment for 11 days with control or with Calc. without or with additional treatment with 1 or 15 µ M ZnSO 4 . (C) Scatterdot plots and arithmetic means±SEM ( n =4, units per milligram protein) of alkaline phosphatase activity in HAoSMCs after treatment for 7 days with control or with β -glycerophosphate (Pi) without or with additional treatment with 1 or 15 µ M ZnSO 4 . (D–F) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of MSX2 (D), CBFA1 (E), and ALPL (F) relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 1 or 15 µ M ZnSO 4 . * P
    Figure Legend Snippet: ZnSO 4 inhibits phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Representative original images showing Alizarin Red staining in HAoSMCs after treatment for 11 days with control or with calcification medium (Calc.) without or with additional treatment with 1 or 15 µ M ZnSO 4 . Images are representative of four independent experiments. The calcified areas are shown as red staining. (B) Scatterdot plots and arithmetic means±SEM ( n =4, micrograms per milligram protein) of calcium content in HAoSMCs after treatment for 11 days with control or with Calc. without or with additional treatment with 1 or 15 µ M ZnSO 4 . (C) Scatterdot plots and arithmetic means±SEM ( n =4, units per milligram protein) of alkaline phosphatase activity in HAoSMCs after treatment for 7 days with control or with β -glycerophosphate (Pi) without or with additional treatment with 1 or 15 µ M ZnSO 4 . (D–F) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of MSX2 (D), CBFA1 (E), and ALPL (F) relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 1 or 15 µ M ZnSO 4 . * P

    Techniques Used: Staining, Activity Assay, Expressing

    ZnSO 4  inhibits phosphate-induced NF- κ B activation and upregulates  TNFAIP3  expression in HAoSMCs. (A) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of normalized phospho-I κ B α  )/I κ B α /GAPDH and total I κ B α /GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with  β -glycerophosphate (Pi) without or with additional treatment with 15  µ M ZnSO 4 . (B) Representative confocal microscopy images showing NF- κ B p65 protein expression and localization in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15  µ M ZnSO 4 . Images are representative for three independent experiments. NF- κ B p65 expression: green labeling, nuclei: purple labeling. Scale bar, 25  μ m. (C) Scatterdot plots and arithmetic means±SEM ( n =4; a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla  constructs and treatment for 24 hours with control or with Pi without or with additional treatment with 15  µ M ZnSO 4 . (D) Scatterdot plots and arithmetic means±SEM ( n =6; a.u.) of  TNFAIP3  relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15  µ M ZnSO 4 . (E) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =5; a.u.) of normalized TNFAIP3/GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15  µ M ZnSO 4 . * P
    Figure Legend Snippet: ZnSO 4 inhibits phosphate-induced NF- κ B activation and upregulates TNFAIP3 expression in HAoSMCs. (A) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of normalized phospho-I κ B α )/I κ B α /GAPDH and total I κ B α /GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B) Representative confocal microscopy images showing NF- κ B p65 protein expression and localization in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . Images are representative for three independent experiments. NF- κ B p65 expression: green labeling, nuclei: purple labeling. Scale bar, 25 μ m. (C) Scatterdot plots and arithmetic means±SEM ( n =4; a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (D) Scatterdot plots and arithmetic means±SEM ( n =6; a.u.) of TNFAIP3 relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Representative original Western blots and scatterdot plots and arithmetic means±SEM ( n =5; a.u.) of normalized TNFAIP3/GAPDH protein ratio in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . * P

    Techniques Used: Activation Assay, Expressing, Western Blot, Confocal Microscopy, Labeling, Activity Assay, Luciferase, Reporter Assay, Transfection, Construct

    Silencing of TNFAIP3 blunts the protective effects of ZnSO 4  on phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla  constructs, silencing for 48 hours with negative control siRNA (Neg.si) or TNFAIP3 siRNA (A20si) and treatment for 24 hours with control or with  β -glycerophosphate (Pi) without or with additional treatment with 15  µ M ZnSO 4 . (B–D) Scatterdot plots and arithmetic means±SEM ( n =8; a.u.) of  MSX2  (B),  CBFA1  (C), and  ALPL  (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with Neg.si or A20si and treatment for 24 hours with control or with Pi without or with additional treatment with 15  µ M ZnSO 4 . (E) Scatterdot plots and arithmetic means±SEM ( n =6, micrograms per milligram protein) of calcium content in HAoSMCs after silencing for 11 days with Neg.si or A20si and treatment with control or with calcification medium (Calc.) without or with additional treatment with 15  µ M ZnSO 4 . * P
    Figure Legend Snippet: Silencing of TNFAIP3 blunts the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of NF- κ B–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs after transfection for 48 hours with NF- κ B–responsive luciferase/ Renilla constructs, silencing for 48 hours with negative control siRNA (Neg.si) or TNFAIP3 siRNA (A20si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . (B–D) Scatterdot plots and arithmetic means±SEM ( n =8; a.u.) of MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with Neg.si or A20si and treatment for 24 hours with control or with Pi without or with additional treatment with 15 µ M ZnSO 4 . (E) Scatterdot plots and arithmetic means±SEM ( n =6, micrograms per milligram protein) of calcium content in HAoSMCs after silencing for 11 days with Neg.si or A20si and treatment with control or with calcification medium (Calc.) without or with additional treatment with 15 µ M ZnSO 4 . * P

    Techniques Used: Activity Assay, Luciferase, Reporter Assay, Transfection, Construct, Negative Control, Expressing

    Silencing of zinc-sensing receptor ZnR/GPR39 blunts the ZnSO 4 -induced  TNFAIP3  expression and the protective effects of ZnSO 4  on phosphate-induced osteoinductive signaling in HAoSMCs. Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of  TNFAIP3  (A),  MSX2  (B),  CBFA1  (C), and  ALPL  (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with negative control siRNA (Neg.si) or GPR39 siRNA (GPR39si) and treatment for 24 hours with control or with  β -glycerophosphate (Pi) without or with additional treatment with 15  µ M ZnSO 4 . ** P
    Figure Legend Snippet: Silencing of zinc-sensing receptor ZnR/GPR39 blunts the ZnSO 4 -induced TNFAIP3 expression and the protective effects of ZnSO 4 on phosphate-induced osteoinductive signaling in HAoSMCs. Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of TNFAIP3 (A), MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs after silencing for 48 hours with negative control siRNA (Neg.si) or GPR39 siRNA (GPR39si) and treatment for 24 hours with control or with β -glycerophosphate (Pi) without or with additional treatment with 15 µ M ZnSO 4 . ** P

    Techniques Used: Expressing, Negative Control

    ZnSO 4  inhibits phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Representative original images showing Alizarin Red staining in HAoSMCs after treatment for 11 days with control or with calcification medium (Calc.) without or with additional treatment with 1 or 15  µ M ZnSO 4 . Images are representative of four independent experiments. The calcified areas are shown as red staining. (B) Scatterdot plots and arithmetic means±SEM ( n =4, micrograms per milligram protein) of calcium content in HAoSMCs after treatment for 11 days with control or with Calc. without or with additional treatment with 1 or 15  µ M ZnSO 4 . (C) Scatterdot plots and arithmetic means±SEM ( n =4, units per milligram protein) of alkaline phosphatase activity in HAoSMCs after treatment for 7 days with control or with  β -glycerophosphate (Pi) without or with additional treatment with 1 or 15  µ M ZnSO 4 . (D–F) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of  MSX2  (D),  CBFA1  (E), and  ALPL  (F) relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 1 or 15  µ M ZnSO 4 . * P
    Figure Legend Snippet: ZnSO 4 inhibits phosphate-induced osteoinductive signaling and calcification in HAoSMCs. (A) Representative original images showing Alizarin Red staining in HAoSMCs after treatment for 11 days with control or with calcification medium (Calc.) without or with additional treatment with 1 or 15 µ M ZnSO 4 . Images are representative of four independent experiments. The calcified areas are shown as red staining. (B) Scatterdot plots and arithmetic means±SEM ( n =4, micrograms per milligram protein) of calcium content in HAoSMCs after treatment for 11 days with control or with Calc. without or with additional treatment with 1 or 15 µ M ZnSO 4 . (C) Scatterdot plots and arithmetic means±SEM ( n =4, units per milligram protein) of alkaline phosphatase activity in HAoSMCs after treatment for 7 days with control or with β -glycerophosphate (Pi) without or with additional treatment with 1 or 15 µ M ZnSO 4 . (D–F) Scatterdot plots and arithmetic means±SEM ( n =6; arbitrary units, a.u.) of MSX2 (D), CBFA1 (E), and ALPL (F) relative mRNA expression in HAoSMCs after treatment for 24 hours with control or with Pi without or with additional treatment with 1 or 15 µ M ZnSO 4 . * P

    Techniques Used: Staining, Activity Assay, Expressing

    32) Product Images from "Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells "

    Article Title: Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells

    Journal: The Journal of Cell Biology

    doi:

    Titration conditions for recombinant CAF-1 to support nucleosome assembly during UV-induced synthesis and competence of various phosphorylated forms in the assay. ( A ) Plasmid DNA, either treated with UV-C at 500 J/m 2 (+) or not treated (−) were incubated in a cytosolic extract in the presence of [α- 32 P] dCTP. Complementation in the supercoiling assay was achieved with increasing amounts of recombinant CAF-1 ( solid triangle ), 0 ng (lanes 2 , 3 , and 4 ), 5 ng (lane 5 ), 10 ng (lane 6 ), 20 ng (lane 7 ), or 40 ng (lane 8 ). Deproteinized DNA was purified and analyzed by agarose gel electrophoresis. The incorporation of radiolabel due to the UV-dependent DNA synthesis was visualized by autoradiography ( Labeled DNA ) and the total population of DNA molecules by ethidium bromide staining of the gel ( Total DNA ). Plasmids were processed as indicated in Materials and Methods. The positions of the supercoiled (form I ), nicked (form Ir ), and closed circular (form II ) forms of plasmid DNA are indicated. Control supercoiled DNA was run in parallel (lane 1 ). ( B ) Recombinant CAF-1 was treated with Lambda phosphatase alone (lane 1 ), in the presence of β-glycerophosphate (lane 2 ) or mock treated (lanes 3 and 5 ), mitotic extract ( M ) and nuclear interphasic extract ( I ) were prepared as described (see Materials and Methods). All samples were processed for Western blot analysis and detection with the polyclonal anti–p60 antibody ( p60 ). Bracket , various phosphorylation forms. ( C ) The three sources of recombinant CAF-1 ( r.CAF-1 ): treated with Lambda phosphatase alone (lane 4 ), in the presence of β-glycerophosphate (lane 5 ), or mock treated (lane 3 ) (shown in B , left ) were used in the assay as described above. The amount added per reaction was 10 ng (defined as the critical amount in A ). The presence and amount of the phosphorylated p60 subunit of CAF-1 (exogenous and endogenous) at the end of each reaction was assessed by Western blotting using the polyclonal anti-p60 antibody ( p60 ). Asterisk , slowest migrating phosphorylated form of p60.
    Figure Legend Snippet: Titration conditions for recombinant CAF-1 to support nucleosome assembly during UV-induced synthesis and competence of various phosphorylated forms in the assay. ( A ) Plasmid DNA, either treated with UV-C at 500 J/m 2 (+) or not treated (−) were incubated in a cytosolic extract in the presence of [α- 32 P] dCTP. Complementation in the supercoiling assay was achieved with increasing amounts of recombinant CAF-1 ( solid triangle ), 0 ng (lanes 2 , 3 , and 4 ), 5 ng (lane 5 ), 10 ng (lane 6 ), 20 ng (lane 7 ), or 40 ng (lane 8 ). Deproteinized DNA was purified and analyzed by agarose gel electrophoresis. The incorporation of radiolabel due to the UV-dependent DNA synthesis was visualized by autoradiography ( Labeled DNA ) and the total population of DNA molecules by ethidium bromide staining of the gel ( Total DNA ). Plasmids were processed as indicated in Materials and Methods. The positions of the supercoiled (form I ), nicked (form Ir ), and closed circular (form II ) forms of plasmid DNA are indicated. Control supercoiled DNA was run in parallel (lane 1 ). ( B ) Recombinant CAF-1 was treated with Lambda phosphatase alone (lane 1 ), in the presence of β-glycerophosphate (lane 2 ) or mock treated (lanes 3 and 5 ), mitotic extract ( M ) and nuclear interphasic extract ( I ) were prepared as described (see Materials and Methods). All samples were processed for Western blot analysis and detection with the polyclonal anti–p60 antibody ( p60 ). Bracket , various phosphorylation forms. ( C ) The three sources of recombinant CAF-1 ( r.CAF-1 ): treated with Lambda phosphatase alone (lane 4 ), in the presence of β-glycerophosphate (lane 5 ), or mock treated (lane 3 ) (shown in B , left ) were used in the assay as described above. The amount added per reaction was 10 ng (defined as the critical amount in A ). The presence and amount of the phosphorylated p60 subunit of CAF-1 (exogenous and endogenous) at the end of each reaction was assessed by Western blotting using the polyclonal anti-p60 antibody ( p60 ). Asterisk , slowest migrating phosphorylated form of p60.

    Techniques Used: Titration, Recombinant, Plasmid Preparation, Incubation, Purification, Agarose Gel Electrophoresis, DNA Synthesis, Autoradiography, Labeling, Staining, Western Blot

    33) Product Images from "In Vitro Long-Term Expansion and High Osteogenic Potential of Periodontal Ligament Stem Cells: More Than a Mirage"

    Article Title: In Vitro Long-Term Expansion and High Osteogenic Potential of Periodontal Ligament Stem Cells: More Than a Mirage

    Journal: Cell Transplantation

    doi: 10.1177/0963689718807680

    Osteogenic differentiation of PDLSCs cultured in EHFM, α-MEM and DMEM enriched with dexamethasone, ascorbic acid and β-glycerophosphate. Figure shows the schema of the experimental set up. Detection of the osteogenic differentiation of EHFM-, α-MEM- and DMEM-cultured PDLSCs in basal conditions and in the presence of osteo-inductive factors through ALP activity and Alizarin Red S staining (A). Relative mRNA expression levels of ALP, OPN, OCN, Runx2, ColI, ColII, IBSP and Msx2 in EHFM-, α-MEM- and DMEM-cultured PDLSCs after differentiation culture for 7, 14 and 21 days (B–D). Relative mRNA expression of all osteogenesis markers in EHFM-expanded PDLSCs at 7, 14 and 21 days was expressed as % of DMEM and α-MEM (E). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; ALP: alkaline phosphatase; Col: collagen; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; IBSP: integrin binding sialoprotein; OCN: osteocalcin; OPN: osteopontin; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.
    Figure Legend Snippet: Osteogenic differentiation of PDLSCs cultured in EHFM, α-MEM and DMEM enriched with dexamethasone, ascorbic acid and β-glycerophosphate. Figure shows the schema of the experimental set up. Detection of the osteogenic differentiation of EHFM-, α-MEM- and DMEM-cultured PDLSCs in basal conditions and in the presence of osteo-inductive factors through ALP activity and Alizarin Red S staining (A). Relative mRNA expression levels of ALP, OPN, OCN, Runx2, ColI, ColII, IBSP and Msx2 in EHFM-, α-MEM- and DMEM-cultured PDLSCs after differentiation culture for 7, 14 and 21 days (B–D). Relative mRNA expression of all osteogenesis markers in EHFM-expanded PDLSCs at 7, 14 and 21 days was expressed as % of DMEM and α-MEM (E). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; ALP: alkaline phosphatase; Col: collagen; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; IBSP: integrin binding sialoprotein; OCN: osteocalcin; OPN: osteopontin; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.

    Techniques Used: Cell Culture, ALP Assay, Activity Assay, Staining, Expressing, Modification, Binding Assay

    34) Product Images from "Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development"

    Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

    Journal: Scientific Reports

    doi: 10.1038/srep46746

    Expression of Klf5 during odontoblastic differentiation of mouse dental papilla mesenchymal cells. iMDP-3 cells were cultured in DM (DMEM supplemented with 10% FBS, antibiotics, 50 μg/mL ascorbic acid, 10 mM sodium β-glycerophosphate and 100 nM dexamethasone) for 0, 1, 3, 5, 7, 11 and 14 days. ( a ) Alizarin Red S (ARS) and ( b ) Alkaline phosphatase (ALP) staining of iMDP-3 cells on days 7 and 14 after differentiation induction by low and high magnifications. ( c ) Cell numbers on days 7 and 14 after differentiation induction. ( d ) Expression of Klf5 protein was detected by Western blot analysis using antibodies specific to Klf5 and β-actin. Protein expression of Klf5 was upregulated during odontoblastic differentiation of iMDP-3 cells. ( e ) Protein expression of Klf5 and β-actin was quantitated using image J software. Expression of Klf5 was normalized to β-actin expression. Expression level of Klf5 proteins on day 0 acts as one-fold increase. ( f ) Klf5, ( g ) Dspp and ( h ) Dmp1 mRNA expression was followed by qRT-PCR relative to Cyclo A. mRNA expression of Klf5, Dspp and Dmp1 is upregulated during odontoblastic differentiation of iMDP-3 cells. Expression level of Klf5, Dsp and Dmp1 at different time periods was divided by the Klf5, Dsp and Dmp1 expressions on day 0. * P
    Figure Legend Snippet: Expression of Klf5 during odontoblastic differentiation of mouse dental papilla mesenchymal cells. iMDP-3 cells were cultured in DM (DMEM supplemented with 10% FBS, antibiotics, 50 μg/mL ascorbic acid, 10 mM sodium β-glycerophosphate and 100 nM dexamethasone) for 0, 1, 3, 5, 7, 11 and 14 days. ( a ) Alizarin Red S (ARS) and ( b ) Alkaline phosphatase (ALP) staining of iMDP-3 cells on days 7 and 14 after differentiation induction by low and high magnifications. ( c ) Cell numbers on days 7 and 14 after differentiation induction. ( d ) Expression of Klf5 protein was detected by Western blot analysis using antibodies specific to Klf5 and β-actin. Protein expression of Klf5 was upregulated during odontoblastic differentiation of iMDP-3 cells. ( e ) Protein expression of Klf5 and β-actin was quantitated using image J software. Expression of Klf5 was normalized to β-actin expression. Expression level of Klf5 proteins on day 0 acts as one-fold increase. ( f ) Klf5, ( g ) Dspp and ( h ) Dmp1 mRNA expression was followed by qRT-PCR relative to Cyclo A. mRNA expression of Klf5, Dspp and Dmp1 is upregulated during odontoblastic differentiation of iMDP-3 cells. Expression level of Klf5, Dsp and Dmp1 at different time periods was divided by the Klf5, Dsp and Dmp1 expressions on day 0. * P

    Techniques Used: Expressing, Cell Culture, ALP Assay, Staining, Western Blot, Software, Quantitative RT-PCR

    35) Product Images from "Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro"

    Article Title: Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro

    Journal: BioMed Research International

    doi: 10.1155/2015/673651

    Multilineage differentiation of porcine DFAT cells. (a) Adipogenic differentiation of DFAT cells at P3, P20, and P50 was induced by MDI (high-glucose DMEM containing 10% FBS, 1 μ M dexamethasone, 500 μ M isobutylmethylxanthine (IBMX), 10 μ g/mL insulin, and 200 μ M indomethacin) for 12 days. Lipid droplets were stained by Oil Red O, bar, 50 μ m. Expression of PPARγ mRNA was assessed by real-time PCR. (b) Osteogenic differentiation of DFAT cells at P3, P20, and P50 was induced by induction medium (high-glucose DMEM containing 10% FBS, 0.1 μ M dexamethasone, 10 mM β -glycerophosphate, and 50 mM ascorbic acid) for 21 days, bar, 200 μ m. Expression of Runx2 mRNA was assessed by real-time PCR. (c) Myogenic differentiation of DFAT cells at P15 and P25 was induced by Galectin-1 for 21 days, expression of myogenic markers was confirmed by immunofluorescence analysis for Desmin and MyHC, and the nuclei were stained by DAPI. Images were merged by Desmin (red) and MyHC (green) with DAPI (blue), bar, 50 μ m. Expression of MyoG mRNA was assessed by real-time PCR. All values are represented as mean ± SD from three independent experiments. ∗ p
    Figure Legend Snippet: Multilineage differentiation of porcine DFAT cells. (a) Adipogenic differentiation of DFAT cells at P3, P20, and P50 was induced by MDI (high-glucose DMEM containing 10% FBS, 1 μ M dexamethasone, 500 μ M isobutylmethylxanthine (IBMX), 10 μ g/mL insulin, and 200 μ M indomethacin) for 12 days. Lipid droplets were stained by Oil Red O, bar, 50 μ m. Expression of PPARγ mRNA was assessed by real-time PCR. (b) Osteogenic differentiation of DFAT cells at P3, P20, and P50 was induced by induction medium (high-glucose DMEM containing 10% FBS, 0.1 μ M dexamethasone, 10 mM β -glycerophosphate, and 50 mM ascorbic acid) for 21 days, bar, 200 μ m. Expression of Runx2 mRNA was assessed by real-time PCR. (c) Myogenic differentiation of DFAT cells at P15 and P25 was induced by Galectin-1 for 21 days, expression of myogenic markers was confirmed by immunofluorescence analysis for Desmin and MyHC, and the nuclei were stained by DAPI. Images were merged by Desmin (red) and MyHC (green) with DAPI (blue), bar, 50 μ m. Expression of MyoG mRNA was assessed by real-time PCR. All values are represented as mean ± SD from three independent experiments. ∗ p

    Techniques Used: Staining, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence

    36) Product Images from "Dentinogenic effects of extracted dentin matrix components digested with matrix metalloproteinases"

    Article Title: Dentinogenic effects of extracted dentin matrix components digested with matrix metalloproteinases

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29112-3

    Effects of 1 µg/ml of MMP-treated dentin matrix components (DMCs) on cell differentiation in rat primary pulp cells. Cells were treated with DMCs treated with matrix metalloproteinase (MMP)-1 ( A , H ), MMP-2 ( B , I ), MMP-3 ( C,J ), MMP-8 ( D,K ), MMP-9 ( D,L ), MMP-13 ( E,M ), and MMP-20 ( G,N ), incubated DMCs without MMP or medium alone containing 50 μg/ml of ascorbic acid and 10 mM of β-glycerophosphate for 7 days ( A–G ) or 14 days ( H–N ). One microgram per millilitre of DMCs treated with MMP-1 ( A ) or MMP-20 ( G ) showed higher alkaline phosphatase (ALP) activities, compared with incubated DMCs without MMPs at 7 days (p
    Figure Legend Snippet: Effects of 1 µg/ml of MMP-treated dentin matrix components (DMCs) on cell differentiation in rat primary pulp cells. Cells were treated with DMCs treated with matrix metalloproteinase (MMP)-1 ( A , H ), MMP-2 ( B , I ), MMP-3 ( C,J ), MMP-8 ( D,K ), MMP-9 ( D,L ), MMP-13 ( E,M ), and MMP-20 ( G,N ), incubated DMCs without MMP or medium alone containing 50 μg/ml of ascorbic acid and 10 mM of β-glycerophosphate for 7 days ( A–G ) or 14 days ( H–N ). One microgram per millilitre of DMCs treated with MMP-1 ( A ) or MMP-20 ( G ) showed higher alkaline phosphatase (ALP) activities, compared with incubated DMCs without MMPs at 7 days (p

    Techniques Used: Cell Differentiation, Incubation, ALP Assay

    Effects of 1 µg/ml of dentin matrix components (DMCs) treated with matrix metalloproteinase (MMP)-1 ( A ), MMP-2 ( B ), MMP-3 ( C ), MMP-8 ( D ), MMP-9 ( E ), MMP-13 ( E ), or MMP-20 ( F ) on mineralized nodule formation in rat primary pulp cells. Cells were exposed to 1 µg/ml of MMP-treated DMCs, incubated DMCs without MMPs or medium alone containing 50 μg/ml of ascorbic acid and 10 mm of β-glycerophosphate for 21 days. DMCs (1 µg/ml) treated with MMP-1 ( A ), -9 ( E ), -13 ( F ), or -20 ( G ). Groups with similar lower-case letters (i.e., a and b) are not significantly different. Data represent five independent experiments.
    Figure Legend Snippet: Effects of 1 µg/ml of dentin matrix components (DMCs) treated with matrix metalloproteinase (MMP)-1 ( A ), MMP-2 ( B ), MMP-3 ( C ), MMP-8 ( D ), MMP-9 ( E ), MMP-13 ( E ), or MMP-20 ( F ) on mineralized nodule formation in rat primary pulp cells. Cells were exposed to 1 µg/ml of MMP-treated DMCs, incubated DMCs without MMPs or medium alone containing 50 μg/ml of ascorbic acid and 10 mm of β-glycerophosphate for 21 days. DMCs (1 µg/ml) treated with MMP-1 ( A ), -9 ( E ), -13 ( F ), or -20 ( G ). Groups with similar lower-case letters (i.e., a and b) are not significantly different. Data represent five independent experiments.

    Techniques Used: Incubation

    37) Product Images from "Fbxl17 is rearranged in breast cancer and loss of its activity leads to increased global O-GlcNAcylation"

    Article Title: Fbxl17 is rearranged in breast cancer and loss of its activity leads to increased global O-GlcNAcylation

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-019-03306-y

    Fbxl17 inhibits the phosphorylation of UAP1. a In vitro GST pull-down assay using bacterially expressed and purified Fbxl17 constructs or Skp2/GST as controls immobilised on a GST column incubated with rabbit reticulocyte lysate (left panel). Fbxl17(321-701aa) and Skp2 constructs contained an IRES_Skp1 to aid expression. Input for rabbit reticulocyte lysate = 20%. Coomassie staining of GST proteins, volume of sample loaded indicated below lanes (right panel) * indicates bands relating to expressed proteins, arrow represents Uap1, n = 2. b HEK293T cells transfected with Fbxl17WT, ΔFbox or empty vector (EV) for 48 h then treated with 10 µM MG132 or DMSO for 4 h. Whole cell lysates immunoblotted with the indicated antibodies, n = 3. c In vivo ubiquitination assay for UAP1. HA-UAP1 immunoprecipitated from HEK293T cells transfected with ubiquitin and indicated Fbxl17 constructs. Membranes probed with anti-UAP1 antibody, arrow indicates modified Uap1, n = 3. d In vivo ubiquitination assay for UAP1 as in c in the presence of β-glycerophosphate (lane 3) and alkaline phosphatase (CIP) (lane 4), n = 2. e LC–MS analysis of total UDP-GlcNAc levels in U2OS cells treated with Fbxl17 siRNA3 or control siRNA for 48 h. Mean ± SEM for five biological replicates, * p
    Figure Legend Snippet: Fbxl17 inhibits the phosphorylation of UAP1. a In vitro GST pull-down assay using bacterially expressed and purified Fbxl17 constructs or Skp2/GST as controls immobilised on a GST column incubated with rabbit reticulocyte lysate (left panel). Fbxl17(321-701aa) and Skp2 constructs contained an IRES_Skp1 to aid expression. Input for rabbit reticulocyte lysate = 20%. Coomassie staining of GST proteins, volume of sample loaded indicated below lanes (right panel) * indicates bands relating to expressed proteins, arrow represents Uap1, n = 2. b HEK293T cells transfected with Fbxl17WT, ΔFbox or empty vector (EV) for 48 h then treated with 10 µM MG132 or DMSO for 4 h. Whole cell lysates immunoblotted with the indicated antibodies, n = 3. c In vivo ubiquitination assay for UAP1. HA-UAP1 immunoprecipitated from HEK293T cells transfected with ubiquitin and indicated Fbxl17 constructs. Membranes probed with anti-UAP1 antibody, arrow indicates modified Uap1, n = 3. d In vivo ubiquitination assay for UAP1 as in c in the presence of β-glycerophosphate (lane 3) and alkaline phosphatase (CIP) (lane 4), n = 2. e LC–MS analysis of total UDP-GlcNAc levels in U2OS cells treated with Fbxl17 siRNA3 or control siRNA for 48 h. Mean ± SEM for five biological replicates, * p

    Techniques Used: In Vitro, Pull Down Assay, Purification, Construct, Incubation, Expressing, Staining, Transfection, Plasmid Preparation, In Vivo, Ubiquitin Assay, Immunoprecipitation, Modification, Liquid Chromatography with Mass Spectroscopy

    38) Product Images from "miR32-5p promoted vascular smooth muscle cell calcification by upregulating TNFα in the microenvironment"

    Article Title: miR32-5p promoted vascular smooth muscle cell calcification by upregulating TNFα in the microenvironment

    Journal: BMC Immunology

    doi: 10.1186/s12865-019-0324-x

    The influence of TNFα expressed by BV2 cells for VSMC calcification. a - f Alizarin red staining analysis of VSMC calcification in differently stimulated condition (40×), a group1 added 10% BV2 culture supernatant and 10 mM β-glycerophosphate, b group3 added 10 mM β-glycerophosphate and 10 ng/ml mouse TNFα, c group5 10 mM β-glycerophosphate, d group2 added 10% BV2 culture supernatant, which was incubated with rabbit anti-mouse TNFα antibody, and 10 mM β-glycerophosphate, e group4 added 10 ng/ml mouse TNFα, f group6 added equal volume PBS. g-i qRT-PCR analysis for the expression of Runx2 and α-SMA in VSMC, g the mRNA expression of Runx2 and α-SMA in groups 1 and 2, h the mRNA expression of Runx2 and α-SMA in groups 3 and 4, i the mRNA expression of Runx2 and α-SMA in groups 5 and 6. Bars represent mean ± S.D. ( n ≥ 3). Significant difference marked with one ( p
    Figure Legend Snippet: The influence of TNFα expressed by BV2 cells for VSMC calcification. a - f Alizarin red staining analysis of VSMC calcification in differently stimulated condition (40×), a group1 added 10% BV2 culture supernatant and 10 mM β-glycerophosphate, b group3 added 10 mM β-glycerophosphate and 10 ng/ml mouse TNFα, c group5 10 mM β-glycerophosphate, d group2 added 10% BV2 culture supernatant, which was incubated with rabbit anti-mouse TNFα antibody, and 10 mM β-glycerophosphate, e group4 added 10 ng/ml mouse TNFα, f group6 added equal volume PBS. g-i qRT-PCR analysis for the expression of Runx2 and α-SMA in VSMC, g the mRNA expression of Runx2 and α-SMA in groups 1 and 2, h the mRNA expression of Runx2 and α-SMA in groups 3 and 4, i the mRNA expression of Runx2 and α-SMA in groups 5 and 6. Bars represent mean ± S.D. ( n ≥ 3). Significant difference marked with one ( p

    Techniques Used: Staining, Incubation, Quantitative RT-PCR, Expressing

    39) Product Images from "In Vitro Long-Term Expansion and High Osteogenic Potential of Periodontal Ligament Stem Cells: More Than a Mirage"

    Article Title: In Vitro Long-Term Expansion and High Osteogenic Potential of Periodontal Ligament Stem Cells: More Than a Mirage

    Journal: Cell Transplantation

    doi: 10.1177/0963689718807680

    Osteogenic differentiation of PDLSCs cultured in EHFM, α-MEM and DMEM enriched with dexamethasone, ascorbic acid and β-glycerophosphate. Figure shows the schema of the experimental set up. Detection of the osteogenic differentiation of EHFM-, α-MEM- and DMEM-cultured PDLSCs in basal conditions and in the presence of osteo-inductive factors through ALP activity and Alizarin Red S staining (A). Relative mRNA expression levels of ALP, OPN, OCN, Runx2, ColI, ColII, IBSP and Msx2 in EHFM-, α-MEM- and DMEM-cultured PDLSCs after differentiation culture for 7, 14 and 21 days (B–D). Relative mRNA expression of all osteogenesis markers in EHFM-expanded PDLSCs at 7, 14 and 21 days was expressed as % of DMEM and α-MEM (E). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; ALP: alkaline phosphatase; Col: collagen; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; IBSP: integrin binding sialoprotein; OCN: osteocalcin; OPN: osteopontin; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.
    Figure Legend Snippet: Osteogenic differentiation of PDLSCs cultured in EHFM, α-MEM and DMEM enriched with dexamethasone, ascorbic acid and β-glycerophosphate. Figure shows the schema of the experimental set up. Detection of the osteogenic differentiation of EHFM-, α-MEM- and DMEM-cultured PDLSCs in basal conditions and in the presence of osteo-inductive factors through ALP activity and Alizarin Red S staining (A). Relative mRNA expression levels of ALP, OPN, OCN, Runx2, ColI, ColII, IBSP and Msx2 in EHFM-, α-MEM- and DMEM-cultured PDLSCs after differentiation culture for 7, 14 and 21 days (B–D). Relative mRNA expression of all osteogenesis markers in EHFM-expanded PDLSCs at 7, 14 and 21 days was expressed as % of DMEM and α-MEM (E). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; ALP: alkaline phosphatase; Col: collagen; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; IBSP: integrin binding sialoprotein; OCN: osteocalcin; OPN: osteopontin; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.

    Techniques Used: Cell Culture, ALP Assay, Activity Assay, Staining, Expressing, Modification, Binding Assay

    40) Product Images from "Ethanol Extracts of Fresh Davallia formosana (WL1101) Inhibit Osteoclast Differentiation by Suppressing RANKL-Induced Nuclear Factor-κB Activation"

    Article Title: Ethanol Extracts of Fresh Davallia formosana (WL1101) Inhibit Osteoclast Differentiation by Suppressing RANKL-Induced Nuclear Factor-κB Activation

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2013/647189

    WL1101 exerts no significant effect on calcium deposition and cell viability in primary cultured rat osteoblasts. (a) For calcium deposition analysis, primary osteoblasts were cultured in 24-well plates and exposed to WL1101 in the presence of L-ascorbic acid and  β -glycerophosphate for 14 days. Long-term treatment with WL1101 exerted no effects on the mineralized deposition of osteoblasts on day 14. The quantitative data are shown in (b) ( n  = 4). (c) For cell viability assay, primary osteoblasts were cultured in 24-well plates and incubated with WL1101 for 2 days. Treatment with WL1101 at 20–200  μ g/mL did not affect cell viability ( n  = 3). Each value represents the mean ± SEM.
    Figure Legend Snippet: WL1101 exerts no significant effect on calcium deposition and cell viability in primary cultured rat osteoblasts. (a) For calcium deposition analysis, primary osteoblasts were cultured in 24-well plates and exposed to WL1101 in the presence of L-ascorbic acid and β -glycerophosphate for 14 days. Long-term treatment with WL1101 exerted no effects on the mineralized deposition of osteoblasts on day 14. The quantitative data are shown in (b) ( n = 4). (c) For cell viability assay, primary osteoblasts were cultured in 24-well plates and incubated with WL1101 for 2 days. Treatment with WL1101 at 20–200  μ g/mL did not affect cell viability ( n = 3). Each value represents the mean ± SEM.

    Techniques Used: Cell Culture, Viability Assay, Incubation

    Related Articles

    MTT Assay:

    Article Title: MSM Enhances GH Signaling via the Jak2/STAT5b Pathway in Osteoblast-Like Cells and Osteoblast Differentiation through the Activation of STAT5b in MSCs
    Article Snippet: .. The anti-phosphotyrosine monoclonal antibody 4G10 and phospho-STAT5b were obtained from Upstate Biotechnology (Lake Placid, NY), The anti-actin antibody , Methyl sulfonemethane (MSM), Alizarin red S, ascorbic acid phosphate, β-glycerophosphate disodium salt hydrate, L-glutamine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, MO). .. The SensoLyte p NPP Alkaline Phosphatase Assay kit was purchased from ANASREC (San Jose, CA).

    Synthesized:

    Article Title: Effect of freeze drying on stability, thermo-responsive characteristics, and in vivo wound healing of erythropoietin-loaded trimethyl chitosan/glycerophosphate hydrogel
    Article Snippet: .. Materials Trimethy chitosan (TMC) with 5% degree of substitution and molecular weight 150-300 kDa was synthesized in our laboratory. β-glycerophosphate disodium salt (GP) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). .. EPO was obtained from Pasteur Institute of Iran (Tehran, I.R.

    Isolation:

    Article Title: Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells
    Article Snippet: .. Protein isolation and western analyses in endothelial cells Cells were washed two times with Hanks solution (Applichem) and lysed with RIPA lysis buffer supplemented with Benzonase (Sigma-Aldrich, Cat# E1014), sodium orthovanadate (Applichem, Cat# A2196), PhosStop (Sigma-Aldrich, Cat# PHOSS-RO), Beta-glycerophosphate (Sigma-Aldrich, Cat# G9422), Sodium fluoride (Sigma-Aldrich, Cat# S7920) for 5–10 min. .. Cell extracts were boiled in Laemmli buffer, equal amounts of protein were separated with SDS-PAGE and gels were blotted onto a nitrocellulose membrane.

    Concentration Assay:

    Article Title: A Three-Dimensional Dense Collagen Hydrogel to Model Cancer Cell/Osteoblast Interactions
    Article Snippet: .. Osteogenic-supplemented medium consisted of alpha-modified MEM (Alpha-MEM; Wisent, St-Bruno, QC, Canada) with the addition of (+)-sodium ʟ-ascorbate (Sigma-Aldrich, Oakville, ON, Canada) to a final concentration of 50 µM, and β-glycerophosphate disodium salt hydrate (Sigma-Aldrich, Canada) to a final concentration of 10 mM. ..

    Lysis:

    Article Title: Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells
    Article Snippet: .. Protein isolation and western analyses in endothelial cells Cells were washed two times with Hanks solution (Applichem) and lysed with RIPA lysis buffer supplemented with Benzonase (Sigma-Aldrich, Cat# E1014), sodium orthovanadate (Applichem, Cat# A2196), PhosStop (Sigma-Aldrich, Cat# PHOSS-RO), Beta-glycerophosphate (Sigma-Aldrich, Cat# G9422), Sodium fluoride (Sigma-Aldrich, Cat# S7920) for 5–10 min. .. Cell extracts were boiled in Laemmli buffer, equal amounts of protein were separated with SDS-PAGE and gels were blotted onto a nitrocellulose membrane.

    Modification:

    Article Title: Differentiation of rhesus adipose stem cells into dopaminergic neurons ☆
    Article Snippet: .. To induce osteogenic differentiation, rhesus adipose stem cells were plated (1 × 105 cells per well) in Corning 6-well plates containing Dulbecco's modified Eagle's medium (high glucose), 10% fetal bovine serum, 10 nM dexamethasone, 10 mM β-glycerophosphate disodium salt hydrate and 150 μM L-ascorbic acid-2-phosphate (Sigma). .. After 3 weeks of osteogenic stimulation, cells were stained with alizarin red[ ].

    Western Blot:

    Article Title: Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells
    Article Snippet: .. Protein isolation and western analyses in endothelial cells Cells were washed two times with Hanks solution (Applichem) and lysed with RIPA lysis buffer supplemented with Benzonase (Sigma-Aldrich, Cat# E1014), sodium orthovanadate (Applichem, Cat# A2196), PhosStop (Sigma-Aldrich, Cat# PHOSS-RO), Beta-glycerophosphate (Sigma-Aldrich, Cat# G9422), Sodium fluoride (Sigma-Aldrich, Cat# S7920) for 5–10 min. .. Cell extracts were boiled in Laemmli buffer, equal amounts of protein were separated with SDS-PAGE and gels were blotted onto a nitrocellulose membrane.

    Molecular Weight:

    Article Title: Effect of freeze drying on stability, thermo-responsive characteristics, and in vivo wound healing of erythropoietin-loaded trimethyl chitosan/glycerophosphate hydrogel
    Article Snippet: .. Materials Trimethy chitosan (TMC) with 5% degree of substitution and molecular weight 150-300 kDa was synthesized in our laboratory. β-glycerophosphate disodium salt (GP) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). .. EPO was obtained from Pasteur Institute of Iran (Tehran, I.R.

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  • 99
    Millipore β glycerophosphate
    AACOCF3 suppressed phosphate-induced calcification and osteogenic/chondrogenic signaling in HAoSMCs. Representative images showing alizarin red staining ( A , n = 3) and scatter dot plots (arithmetic mean ± SEM) of calcium content ( B , n = 4, μg/mg protein) in HAoSMCs following treatment with control (CTR) or with calcification medium (Calc.), without or with additional treatment with 10 μM AACOCF3 (AAC). The calcified areas are shown as red staining. ( C – G ) Scatter dot plots (arithmetic mean ± SEM) of MSX2 ( C ), CBFA1 ( D ), and ALPL ( E ) relative mRNA expression ( n = 6; arbitrary units [a.u.]) and ALPL activity ( F , n = 4, U/mg protein) in HAoSMCs as well as arachidonic acid (AA) levels ( G , n = 4; ng/mg protein) in the cell culture medium of HAoSMCs following treatment with control or with <t>β-glycerophosphate</t> (Pi), without or with additional treatment with 10 μM AACOCF3. * P
    β Glycerophosphate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 331 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β glycerophosphate/product/Millipore
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    84
    Millipore lats1 kinase buffer
    Experimental validation of model predictions. A) HEK293 cells were transfected with non targeted siRNA (Scr) of the indicated siRNA against <t>LATS1.</t> Phosphorylation of CREB or p53 was measured using specific antibodies and normalised to the level of expression of the corresponding proteins. The graph shows the fold change of the phosphorylation of the specific residues with respect to the Scr control. B) HEK293 were transfected with empty vector (EV) or GAG-AKT or treated with AKTi IV (10M) for 1 hour. Phosphorylated proteins were immunoprecipitated using an anti-AKT antibody and the immunoprecipitates were blotted with anti-MST2. The bars show the fold change with respect to the control. The experiments were repeated at least 2 times. Error bars represent standard variations.
    Lats1 Kinase Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    AACOCF3 suppressed phosphate-induced calcification and osteogenic/chondrogenic signaling in HAoSMCs. Representative images showing alizarin red staining ( A , n = 3) and scatter dot plots (arithmetic mean ± SEM) of calcium content ( B , n = 4, μg/mg protein) in HAoSMCs following treatment with control (CTR) or with calcification medium (Calc.), without or with additional treatment with 10 μM AACOCF3 (AAC). The calcified areas are shown as red staining. ( C – G ) Scatter dot plots (arithmetic mean ± SEM) of MSX2 ( C ), CBFA1 ( D ), and ALPL ( E ) relative mRNA expression ( n = 6; arbitrary units [a.u.]) and ALPL activity ( F , n = 4, U/mg protein) in HAoSMCs as well as arachidonic acid (AA) levels ( G , n = 4; ng/mg protein) in the cell culture medium of HAoSMCs following treatment with control or with β-glycerophosphate (Pi), without or with additional treatment with 10 μM AACOCF3. * P

    Journal: JCI Insight

    Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

    doi: 10.1172/jci.insight.125638

    Figure Lengend Snippet: AACOCF3 suppressed phosphate-induced calcification and osteogenic/chondrogenic signaling in HAoSMCs. Representative images showing alizarin red staining ( A , n = 3) and scatter dot plots (arithmetic mean ± SEM) of calcium content ( B , n = 4, μg/mg protein) in HAoSMCs following treatment with control (CTR) or with calcification medium (Calc.), without or with additional treatment with 10 μM AACOCF3 (AAC). The calcified areas are shown as red staining. ( C – G ) Scatter dot plots (arithmetic mean ± SEM) of MSX2 ( C ), CBFA1 ( D ), and ALPL ( E ) relative mRNA expression ( n = 6; arbitrary units [a.u.]) and ALPL activity ( F , n = 4, U/mg protein) in HAoSMCs as well as arachidonic acid (AA) levels ( G , n = 4; ng/mg protein) in the cell culture medium of HAoSMCs following treatment with control or with β-glycerophosphate (Pi), without or with additional treatment with 10 μM AACOCF3. * P

    Article Snippet: HAoSMCs were treated for 24 hours (qRT-PCR, AA release), 7 days (ALPL assay), or 11 days (calcification) with 2 mM β-glycerophosphate (MilliporeSigma) ( , , , , ) or with 10 μM AACOCF3.

    Techniques: Staining, Expressing, Activity Assay, Cell Culture

    Silencing of cPLA2 inhibited phosphate-induced osteogenic/chondrogenic signaling in HAoSMCs. Scatter dot plots (arithmetic mean ± SEM) ( n = 6, arbitrary units [a.u.]) of PLA2G4A ( A ), MSX2 ( B ), CBFA1 ( C ), and ALPL ( D ) relative mRNA expression in HAoSMCs following silencing with negative control siRNA (Neg.si) or cPLA2 siRNA (cPLA2si), without or with additional treatment with β-glycerophosphate (Pi). * P

    Journal: JCI Insight

    Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

    doi: 10.1172/jci.insight.125638

    Figure Lengend Snippet: Silencing of cPLA2 inhibited phosphate-induced osteogenic/chondrogenic signaling in HAoSMCs. Scatter dot plots (arithmetic mean ± SEM) ( n = 6, arbitrary units [a.u.]) of PLA2G4A ( A ), MSX2 ( B ), CBFA1 ( C ), and ALPL ( D ) relative mRNA expression in HAoSMCs following silencing with negative control siRNA (Neg.si) or cPLA2 siRNA (cPLA2si), without or with additional treatment with β-glycerophosphate (Pi). * P

    Article Snippet: HAoSMCs were treated for 24 hours (qRT-PCR, AA release), 7 days (ALPL assay), or 11 days (calcification) with 2 mM β-glycerophosphate (MilliporeSigma) ( , , , , ) or with 10 μM AACOCF3.

    Techniques: Expressing, Negative Control

    Knockdown of ATP6V1H upregulates the Akt/GSK3β signaling pathway in MC3T3-E1 cells. (A) The expression of ATP6V1H was detected by western blotting of protein samples from high glucose cultures of MC3T3-E1 cells transfected with si-NC or si-ATP6V1H. (B) Quantitative assay of ATP6V1H western blot was performed by Image J software. (C) The expression of p -Akt, Akt, p -GSK3β and GSK3β was detected by western blotting of protein samples from high glucose cultures of MC3T3-E1 cells transfected with si-NC or si-ATP6V1H. (D) Quantitative assay was performed by Image J software. OM, osteogenic medium; HG, high glucose-free fatty acids; sh-NC, negative control siRNA; si-ATP6V1H, ATP6V1H siRNA; Akt, Serine/Threonine Kinase 1; GSK-3β, Glycogen Synthase Kinase 3 beta. *P

    Journal: Organogenesis

    Article Title: ATP6V1H facilitates osteogenic differentiation in MC3T3-E1 cells via Akt/GSK3β signaling pathway

    doi: 10.1080/15476278.2019.1633869

    Figure Lengend Snippet: Knockdown of ATP6V1H upregulates the Akt/GSK3β signaling pathway in MC3T3-E1 cells. (A) The expression of ATP6V1H was detected by western blotting of protein samples from high glucose cultures of MC3T3-E1 cells transfected with si-NC or si-ATP6V1H. (B) Quantitative assay of ATP6V1H western blot was performed by Image J software. (C) The expression of p -Akt, Akt, p -GSK3β and GSK3β was detected by western blotting of protein samples from high glucose cultures of MC3T3-E1 cells transfected with si-NC or si-ATP6V1H. (D) Quantitative assay was performed by Image J software. OM, osteogenic medium; HG, high glucose-free fatty acids; sh-NC, negative control siRNA; si-ATP6V1H, ATP6V1H siRNA; Akt, Serine/Threonine Kinase 1; GSK-3β, Glycogen Synthase Kinase 3 beta. *P

    Article Snippet: To induce osteogenic differentiation, MC3T3-E1 cells were cultured in α-MEM with10% FBS, 20 mM beta-glycerophosphate and 100 mg/mL ascorbic acid for 14 days.

    Techniques: Expressing, Western Blot, Transfection, Software, Negative Control

    Experimental validation of model predictions. A) HEK293 cells were transfected with non targeted siRNA (Scr) of the indicated siRNA against LATS1. Phosphorylation of CREB or p53 was measured using specific antibodies and normalised to the level of expression of the corresponding proteins. The graph shows the fold change of the phosphorylation of the specific residues with respect to the Scr control. B) HEK293 were transfected with empty vector (EV) or GAG-AKT or treated with AKTi IV (10M) for 1 hour. Phosphorylated proteins were immunoprecipitated using an anti-AKT antibody and the immunoprecipitates were blotted with anti-MST2. The bars show the fold change with respect to the control. The experiments were repeated at least 2 times. Error bars represent standard variations.

    Journal: bioRxiv

    Article Title: Accurate Prediction of Kinase-Substrate Networks Using Knowledge Graphs

    doi: 10.1101/865055

    Figure Lengend Snippet: Experimental validation of model predictions. A) HEK293 cells were transfected with non targeted siRNA (Scr) of the indicated siRNA against LATS1. Phosphorylation of CREB or p53 was measured using specific antibodies and normalised to the level of expression of the corresponding proteins. The graph shows the fold change of the phosphorylation of the specific residues with respect to the Scr control. B) HEK293 were transfected with empty vector (EV) or GAG-AKT or treated with AKTi IV (10M) for 1 hour. Phosphorylated proteins were immunoprecipitated using an anti-AKT antibody and the immunoprecipitates were blotted with anti-MST2. The bars show the fold change with respect to the control. The experiments were repeated at least 2 times. Error bars represent standard variations.

    Article Snippet: Sample were diluted down with 2 ml of LATS1 kinase buffer (25 mM HEPES pH 7.4, 50 mM NaCl, 5 mM MgCl2 and 5 mM MnCl2, 5 mM β -glycerophosphate and 1 mM dithiothreitol) and desalted using a Millipore Amicon ultrafiltration columns with a 3 kDa molecular weight cutoff.

    Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

    Mass-spectrometry validation of a subset of LinkPhinder predicted phosphorylations. Raw intensity values (dots) of the detected phosphorylation sites in GFP-LATS1 associated proteins under the indicated conditions (n=6 replicates), and corresponding box plots indicating median (red line), upper and lower quartile (grey box), whiskers (most extreme values not defined as outliers), and outliers (plus marks) defined as values outside 1.5 times the interquartile range.

    Journal: bioRxiv

    Article Title: Accurate Prediction of Kinase-Substrate Networks Using Knowledge Graphs

    doi: 10.1101/865055

    Figure Lengend Snippet: Mass-spectrometry validation of a subset of LinkPhinder predicted phosphorylations. Raw intensity values (dots) of the detected phosphorylation sites in GFP-LATS1 associated proteins under the indicated conditions (n=6 replicates), and corresponding box plots indicating median (red line), upper and lower quartile (grey box), whiskers (most extreme values not defined as outliers), and outliers (plus marks) defined as values outside 1.5 times the interquartile range.

    Article Snippet: Sample were diluted down with 2 ml of LATS1 kinase buffer (25 mM HEPES pH 7.4, 50 mM NaCl, 5 mM MgCl2 and 5 mM MnCl2, 5 mM β -glycerophosphate and 1 mM dithiothreitol) and desalted using a Millipore Amicon ultrafiltration columns with a 3 kDa molecular weight cutoff.

    Techniques: Mass Spectrometry