Journal: Molecular medicine reports

Article Title: MicroRNA‑125b suppresses the proliferation and osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells.

doi: 10.3892/mmr.2014.2024

Figure Lengend Snippet: Figure 1. Comparison of the proliferation and osteogenic differen tiation of hBMSCs isolated from osteoporotic patients and normal subjects. (A) Growth and viability of hBMSCs were determined by the MTT assay after cells were cultured for 2, 4, 6, 8 and 10 days; (B) hBMSCs at passage 3 were cultured in osteogenic medium for 14 days, followed by staining and assessment of ALP activity. Data were analyzed using the Student's t‑tests; (C) quantitative polymerase chain reaction analysis of miR‑125b expression in hBMSCs. miR‑125b expression levels were increased in hBMSCs isolated from elderly patients. Data were subjected to Student's t‑tests. Error bars represent the mean ± standard deviation of three independent experiments. *P<0.05. hBMSCs, human bone marrow‑derived mesenchymal stem cells; ALP, alkaline phosphatase; miR, micro RNA; OD, optical density.

Article Snippet: On every third day, the medium was replaced with osteogenic differentiation medium (10% fetal bovine serum, Hyclone; 100 nM dexamethasone, 45 mM L-ascorbic acid and 10 mM β-glycerophosphate; Sigma, St. Louis, MO, USA).

Techniques: Comparison, Isolation, MTT Assay, Cell Culture, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

Journal: Molecular medicine reports

Article Title: MicroRNA‑125b suppresses the proliferation and osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells.

doi: 10.3892/mmr.2014.2024

Figure Lengend Snippet: Figure 3. (A) Overexpression of miR‑125b suppressed the osteogenic differentiation of hBMSCs. Osteoblast differentiation of hBMSCs was induced by osteogenic differentiation medium and hBMSCs were collected 14 days after osteogenic induction. qPCR analysis measured the expression of Runx‑2, ALP, OC and COL1α1 in hBMSCs. qPCR data were subjected to Student's t‑tests. Error bars represent the mean ± standard deviation of three independent experi ments. *P<0.05. (B) ALP staining was performed after 14 days of culture to detect ALP activity and Alizarin Red staining was performed after 21 days to evaluate mineralized bone matrix formation. (C) ALP activity of hBMSCs was determined 14 days after osteogenic induction by p‑Nitrophenyl Phosphate Liquid Substrate System and absorbance was measured at 405 nm. Data were subjected to Student's t‑test. *P<0.05. hBMSCs, human bone marrow‑derived mesenchymal stem cells; qPCR, quantitative polymerase chain reaction; Runx‑2, Runt‑related transcription factor‑2; ALP, alkaline phosphatase; OC, osteo calcin; COL1α1, collagen type I α1; NC, negative control; miR, micro RNA; OD, optical density.

Article Snippet: On every third day, the medium was replaced with osteogenic differentiation medium (10% fetal bovine serum, Hyclone; 100 nM dexamethasone, 45 mM L-ascorbic acid and 10 mM β-glycerophosphate; Sigma, St. Louis, MO, USA).

Techniques: Over Expression, Expressing, Standard Deviation, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Negative Control

Journal: Dose-Response

Article Title: Skeletal Stem Cells Rescue Radiation-Induced Osteogenic Precursor Cell Dysfunction via the Wnt/β-Catenin Signaling Pathway

doi: 10.1177/15593258261440983

Figure Lengend Snippet: Co-culture with SSCs rescues the function of irradiated osteogenic precursor cells. (A, B) Cell apoptosis was analyzed by flow cytometry with Annexin V-PE/7AAD double staining. (A) Representative flow cytometry plots. (B) Quantitative analysis of the apoptotic rate. (C, D) ALP activity was assessed. (C) Representative ALP staining images (Scale bar: 50 μm). (D) Quantitative analysis of the relative ALP activity. (E, F) Mineralization capacity was evaluated using Alizarin Red S staining. (E) Representative staining images of mineralized nodules (Scale bar: 100 μm). (F) Quantitative analysis of the relative mineralization level. (G, H) Cell migration was determined by a migration assay. (G) Representative images of migrated cells (Scale bar: 50 μm). (H) Quantitative analysis of the relative cell migration level. All data are presented as mean ± SD, with statistical significance determined by unpaired two-tailed Student’s t-test (* p < 0.05; ** p < 0.01).

Article Snippet: After irradiation and corresponding interventions, cells were cultured in osteogenic induction medium (iXCells Biotechnologies, San Diego, CA, Cat. No. MD-0006) for 7 days.

Techniques: Co-Culture Assay, Irradiation, Flow Cytometry, Double Staining, Activity Assay, Staining, Migration, Two Tailed Test

Journal: Dose-Response

Article Title: Skeletal Stem Cells Rescue Radiation-Induced Osteogenic Precursor Cell Dysfunction via the Wnt/β-Catenin Signaling Pathway

doi: 10.1177/15593258261440983

Figure Lengend Snippet: SSCs exert rescue effects via the Wnt/β-catenin signaling pathway. (A) Representative ALP staining images of cells in each group (Scale bar: 50 μm). (B) Quantitative analysis of ALP activity in each group. (C) Representative Alizarin Red S staining images of cells in each group (Scale bar: 100 μm). (D) Quantitative analysis of Alizarin Red S staining in each group. (E) Relative mRNA expression levels of osteogenic marker genes ( Runx2 , Col1a1 , and OCN ) detected by qRT-PCR. GAPDH was used as an internal reference gene. (F) Representative Western blot images showing the expression levels of RUNX2, COL1A1, OCN, and β-catenin in each group. GAPDH was used as a loading control. (G) Quantitative analysis of Western blot results (gray value ratio of target protein to GAPDH) in each group. All data are presented as mean ± SD, with statistical significance determined by unpaired two-tailed Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: After irradiation and corresponding interventions, cells were cultured in osteogenic induction medium (iXCells Biotechnologies, San Diego, CA, Cat. No. MD-0006) for 7 days.

Techniques: Staining, Activity Assay, Expressing, Marker, Quantitative RT-PCR, Western Blot, Control, Two Tailed Test

Journal: Dose-Response

Article Title: Skeletal Stem Cells Rescue Radiation-Induced Osteogenic Precursor Cell Dysfunction via the Wnt/β-Catenin Signaling Pathway

doi: 10.1177/15593258261440983

Figure Lengend Snippet: SSCs alleviate the radiation-induced bone injury in mice. (A–G) Micro-CT analysis of bone microstructure. (A) Representative micro-CT images of femurs. Quantitative analysis of (B) bone mineral density (BMD), (C) bone volume fraction (BV/TV), (D) trabecular thickness (Tb.Th), (E) trabecular number (Tb.N), (F) connectivity density (Conn.D), and (G) trabecular separation (Tb.Sp) at 2- and 4-weeks post irradiation. (H–K) Histological analysis (Scale bar: 100 μm). (H) H&E staining showing steatosis (arrows) and (I) quantitative analysis of steatotic lesions per field. (J) TRAP staining showing osteoclasts (arrows) and (K) quantitative analysis of osteoclast number per field. (L–O) Immunohistochemical staining of osteogenic markers (Scale bar: 100 μm). (L) Osterix staining and (M) quantitative analysis of Osterix-positive area. (N) β-catenin staining and (O) quantitative analysis of β-catenin-positive area. All experiments were conducted in three groups: Control, irradiation (IR), and IR plus SSC (IR+SSC) at 2- and 4-weeks post-irradiation. All data are presented as mean ± SD, with statistical significance determined by unpaired two-tailed Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: After irradiation and corresponding interventions, cells were cultured in osteogenic induction medium (iXCells Biotechnologies, San Diego, CA, Cat. No. MD-0006) for 7 days.

Techniques: Micro-CT, Irradiation, Staining, Immunohistochemical staining, Control, Two Tailed Test

Journal: Cells

Article Title: Melatonin Attenuates Spinal Cord Injury in Mice by Activating the Nrf2/ARE Signaling Pathway to Inhibit the NLRP3 Inflammasome

doi: 10.3390/cells11182809

Figure Lengend Snippet: The oxidative damage scavenging effect of melatonin in neuron cells. ( A ) The survival rates of PC12 cells that received different concentrations of melatonin treatment were detected using a CCK-8 kit (n = 8). ( B ) The ROS produced in PC12 cells that received different treatments were detected by ROS staining (n = 6, scale bar = 100 µm). ( C , D ) The mitochondrial membrane potential in PC12 cells treated with drugs was detected by JC-1 staining (n = 8, scale bar = 100 µm). ( E – G ) The contents of SOD and MDA and the activity of GSH-PX were detected using the corresponding kits in PC12 cells treated with different treatment factors (n = 10). Data are shown as the means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-18 were measured in the PC12 cell culture medium using ELISA kits according to the manufacturer’s instructions (Elabscience, China).

Techniques: CCK-8 Assay, Produced, Staining, Membrane, Activity Assay

Journal: Cells

Article Title: Melatonin Attenuates Spinal Cord Injury in Mice by Activating the Nrf2/ARE Signaling Pathway to Inhibit the NLRP3 Inflammasome

doi: 10.3390/cells11182809

Figure Lengend Snippet: Melatonin suppresses the NLRP3 inflammasome in PC12 cells treated with H2O2 by activating the Nrf2/ARE pathway. ( A – I ) Western blot detection and statistical analysis of NLRP3-inflammasome-related proteins NLRP3, caspase-1, ASC, and IL-1β; and antioxidant-stress-related proteins nuclear Nrf2, HO-1, and NQO-1 in PC12 cells from each group (n = 6). ( J , K ) The levels of IL-1β and IL-18 in the supernatant of PC12 cells were determined by ELISA kits (n = 6). Data are shown as the means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-18 were measured in the PC12 cell culture medium using ELISA kits according to the manufacturer’s instructions (Elabscience, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Melatonin Attenuates Spinal Cord Injury in Mice by Activating the Nrf2/ARE Signaling Pathway to Inhibit the NLRP3 Inflammasome

doi: 10.3390/cells11182809

Figure Lengend Snippet: Immunofluorescence double staining of Nrf2 and NLRP3 in PC12 cells treated with H2O2. Immunofluorescence double staining was performed to test the protein level of Nrf2 ( A , B ) and NLRP3 ( C , D ) in PC12 cells from each group (n = 8, scale bar = 100 µm). Data are shown as the means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-18 were measured in the PC12 cell culture medium using ELISA kits according to the manufacturer’s instructions (Elabscience, China).

Techniques: Immunofluorescence, Double Staining

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression of Matrix Metalloproteinase Gene Expression by the Orphan Nuclear Receptor NURR1 in Cartilage

doi: 10.1074/jbc.m608327200

Figure Lengend Snippet: FIGURE2.PGE2isapotentinducerofNURR1inchondrocytes.A,primaryhumanchondrocytesderivedfrom patients with OA were treated with PGE2 (10 M) for the times indicated. Nur77, NURR1, and NOR-1 mRNAs were measured by real-time RT-PCR, and levels were normalized to GAPDH. Results are representative of expression profiles from chondrocytes derived from five different patients. B, SW1353 cells were treated in triplicate with PGE2 (1 M) for the times indicated (hours). Nur77, NURR1, and NOR-1 mRNAs were measured by real-time RT-PCR, and levels were normalized to GAPDH. C, nuclear extracts were generated from SW1353 cells treated with PGE2 (1 M) for the times indicated. Western blotting was conducted with anti-NURR1 polyclonal antibody. In vitro translated NURR1 is shown as a positive control. Untr, untreated.

Article Snippet: Human chondrocytes were maintained in chondrocyte growth medium (Cell Applications) as monolayer cultures for not more than 3 days to maintain the chondrocyte phenotype.

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Generated, Western Blot, In Vitro, Positive Control