Journal: Molecular Medicine Reports

Article Title: Platelet-rich plasma protects rat chondrocytes from interleukin-1β-induced apoptosis

doi: 10.3892/mmr.2016.5767

Figure Lengend Snippet: List of antibodies used in western blotting.

Article Snippet: Permeabilization and blocking were performed by incubating the cells with 1% Triton-X 100 and 1% bovine serum albumin for 15 min. Cover slips were then incubated overnight at 4°C with rabbit polyclonal anti-COL2 primary antibodies (cat. no. Bs-11929R; dilution, 1:500; Bioss, Inc., Woburn, MA, USA).

Techniques: Western Blot

Journal: Molecular Medicine Reports

Article Title: Platelet-rich plasma protects rat chondrocytes from interleukin-1β-induced apoptosis

doi: 10.3892/mmr.2016.5767

Figure Lengend Snippet: Platelet-rich plasma (PRP) inhibits the expression of catabolic genes induced by interleukin (IL)-1β. (A) Quantitative polymerase chain reaction was performed 24 h following treatment of chondrocytes (N=3). (B) Western blotting detected downregulation of matrix metalloproteinases (MMPs), and upregulation of tissue inhibitor of metalloproteinases (TIMP), SRY-box 9 (SOX9) and collagen type II (COL2) in the IL-1β + PRP group compared with the IL-1β group. (C) Semi-quantification of blots confirmed the expression of anabolic proteins was increased by PRP (N=3). *P<0.05, vs. the IL-1β group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Permeabilization and blocking were performed by incubating the cells with 1% Triton-X 100 and 1% bovine serum albumin for 15 min. Cover slips were then incubated overnight at 4°C with rabbit polyclonal anti-COL2 primary antibodies (cat. no. Bs-11929R; dilution, 1:500; Bioss, Inc., Woburn, MA, USA).

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

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes

doi: 10.1186/s13287-020-01909-y

Figure Lengend Snippet: Primers of qPCR

Article Snippet: The membrane was blotted with antibodies against COL2 (1:1000, Abcam, USA), SOX9 (1:2000, Sigma-Aldrich), RUNX2 (1:500, Sigma-Aldrich), MMP13 (1:1000, Abcam), CBFB (1:1000, Santa Cruz Biotechnology, USA), COL10 (1:2000, Abcam), β-catenin (1:1000, CST, USA), PPARγ (1:1000, CST, USA), and GADPH (1:2000, TransGen) at 4 °C overnight, followed by incubation with peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG antibody (1:2000, TransGen) at room temperature for 60 min.

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes

doi: 10.1186/s13287-020-01909-y

Figure Lengend Snippet: Confirmation of chondrogenic differentiation of hBMSC chondrogenic pellets. a mRNA expression of COL2 , SOX9 , COL10 , RUNX2 , and CBFB at days 0, 7, 14, and 21, as well as in control chondrocytes (chon) as measured by RT-qPCR. * P < 0.05, ** P < 0.001. b Western blot analysis of expression of COL2, SOX9, COL10, RUNX2, and CBFB in hBMSC-derived cartilage pellets on 0, 7, 14, and 21 days. c Semi-quantification of western blot data. * P < 0.05, ** P < 0.001. d IHC for COL2, SOX9, and COL10 in cartilage pellets on 7, 14, and 21 days. Scale bar = 50 μm. e Optical density for COL2, SOX9, and COL10 was evaluated by ImageJ software, and the data represented the expression levels of COL2, SOX9, and COL10. * P < 0.05, ** P < 0.001

Article Snippet: The membrane was blotted with antibodies against COL2 (1:1000, Abcam, USA), SOX9 (1:2000, Sigma-Aldrich), RUNX2 (1:500, Sigma-Aldrich), MMP13 (1:1000, Abcam), CBFB (1:1000, Santa Cruz Biotechnology, USA), COL10 (1:2000, Abcam), β-catenin (1:1000, CST, USA), PPARγ (1:1000, CST, USA), and GADPH (1:2000, TransGen) at 4 °C overnight, followed by incubation with peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG antibody (1:2000, TransGen) at room temperature for 60 min.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Software

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes

doi: 10.1186/s13287-020-01909-y

Figure Lengend Snippet: miR-27b overexpression inhibits hypertrophic differentiation of hBMSC-derived chondrocytes. a Expression of COL2, SOX9, RUNX2, CBFB, MMP13, and PPARγ upon overexpression of miR-27b mimic in hBMSCs as measured by RT-qPCR on day 3. * P < 0.05, ** P < 0.001. b Western blot measurement of expression of COL2, SOX9, RUNX2, CBFB, MMP13, and PPARγ expression in miR-27b-overexpressing hBMSCs compared to NC on day 3 after transfection. c Semi-quantification of western blot data. * P < 0.05, ** P < 0.001. d miR-27b expression on day 0 after miR-27b-LV and miR-27b-SC-LV transfection followed by chondrogenic induction, as measured by RT-qPCR. * P < 0.05. e miR-27b expression on day 14 after miR-27b-LV and miR-27b-SC-LV transfection followed by chondrogenic induction for 14 days, as measured by RT-qPCR. ** P < 0.001. f Safranin O staining and IHC for COL2, SOX9, and COL10 in cartilage pellets on day 14. Scale bar = 20 μm. g Safranin O staining was performed to measure GAGs. Optical density for GAGs was evaluated by ImageJ software, and the data represented GAG content. Optical density for COL2, SOX9, and COL10 was evaluated by ImageJ software, and the data represented the expression levels of COL2, SOX9, and COL10. * P < 0.05

Article Snippet: The membrane was blotted with antibodies against COL2 (1:1000, Abcam, USA), SOX9 (1:2000, Sigma-Aldrich), RUNX2 (1:500, Sigma-Aldrich), MMP13 (1:1000, Abcam), CBFB (1:1000, Santa Cruz Biotechnology, USA), COL10 (1:2000, Abcam), β-catenin (1:1000, CST, USA), PPARγ (1:1000, CST, USA), and GADPH (1:2000, TransGen) at 4 °C overnight, followed by incubation with peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG antibody (1:2000, TransGen) at room temperature for 60 min.

Techniques: Over Expression, Derivative Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Staining, Software

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes

doi: 10.1186/s13287-020-01909-y

Figure Lengend Snippet: miR-27b inhibits hypertrophic differentiation of hBMSC-derived chondrocytes and upregulates SOX9 expression. a Protein levels of CBFB and RUNX2 in coimmunoprecipitates. b After sh-CBFB and sh-SC transfection, on 14 days mRNA expression of CBFB , RUNX2 , COL10 , SOX9 , and COL2 as measured by RT-qPCR. * P < 0.05, ** P < 0.001. c Protein levels of CBFB, RUNX2, SOX9, and COL10 as measured by western blot. d Semi-quantification of western blot data. * P < 0.05, ** P < 0.001. e IHC staining of COL2, SOX9, and COL10 expression in chondrogenic pellets transfected with NC, sh-SC, and sh-CBFB. Scale bar = 20 μm. f Measurement of optical density for COL 2, SOX9, and COL10 was evaluated by ImageJ software, and the data represented the expression levels of COL 2, SOX9, and COL10. * P < 0.05

Article Snippet: The membrane was blotted with antibodies against COL2 (1:1000, Abcam, USA), SOX9 (1:2000, Sigma-Aldrich), RUNX2 (1:500, Sigma-Aldrich), MMP13 (1:1000, Abcam), CBFB (1:1000, Santa Cruz Biotechnology, USA), COL10 (1:2000, Abcam), β-catenin (1:1000, CST, USA), PPARγ (1:1000, CST, USA), and GADPH (1:2000, TransGen) at 4 °C overnight, followed by incubation with peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG antibody (1:2000, TransGen) at room temperature for 60 min.

Techniques: Derivative Assay, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Software

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes

doi: 10.1186/s13287-020-01909-y

Figure Lengend Snippet: miR-27b overexpression in hBMSCs inhibits their hypertrophic differentiation and enhances their ability to repair cartilage defects in a rat model. a Gross photos of articular cartilage defects in a rat model. Cartilage pellets induced from hBMSCs transfected with NC, miR-27b-SC-LV, and miR-27b-LV group ( n = 6 in each group) were transplanted into model rats with full-thickness articular cartilage lesions. HE, Toluidine blue, and Safranin O staining of femoral sections. Scale bar = 100 μm. b Safranin O staining and Toluidine blue staining were performed to measure levels of GAGs and proteoglycans. Optical density for GAGs and proteoglycans was evaluated by ImageJ software, and the data represented GAG and proteoglycan content. c IHC staining of human nuclei, COL1, COL2, and COL10 in femoral sections to evaluate repair efficacy. Scale bar = 100 μm. d Optical density for human nuclei, COL1, COL2, and COL10 was evaluated by ImageJ software, and the data represented the expression levels of human nuclei, COL1, COL2, and COL10. * P < 0.05, ** P < 0.001. e Mean histological scores obtained using the O’Driscoll score for cartilage repair at 4 weeks. * P < 0.05

Article Snippet: The membrane was blotted with antibodies against COL2 (1:1000, Abcam, USA), SOX9 (1:2000, Sigma-Aldrich), RUNX2 (1:500, Sigma-Aldrich), MMP13 (1:1000, Abcam), CBFB (1:1000, Santa Cruz Biotechnology, USA), COL10 (1:2000, Abcam), β-catenin (1:1000, CST, USA), PPARγ (1:1000, CST, USA), and GADPH (1:2000, TransGen) at 4 °C overnight, followed by incubation with peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG antibody (1:2000, TransGen) at room temperature for 60 min.

Techniques: Over Expression, Transfection, Staining, Software, Immunohistochemistry, Expressing

Journal: Cell Transplantation

Article Title: Laryngeal Cartilage Regeneration of Nude Rats by Transplantation of Mesenchymal Stem Cells Derived from Human-Induced Pluripotent Stem Cells

doi: 10.1177/09636897231178460

Figure Lengend Snippet: Immunofluorescence staining of cartilage-like tissue regenerated tissue using anti-SOX9, HNA, type II collagen (COL2), type I collagen (COL1) antibody, and DAPI staining. (A) The area between the yellow arrowheads indicates the defect area in the thyroid cartilage. (B–G) Higher magnification of the yellow box in (A). (B) SOX9; (C) HNA; (D) DAPI; (E) merge of SOX9 and HNA; (F) merge of COL2 (green), HNA (red), and DAPI (blue); (G) merge of COL1 (white), HNA (red), and DAPI (blue). (A) Scale bar = 200 μm, (B–G): 50 μm.

Article Snippet: Sections were incubated overnight at 4°C with anti-human nuclear antigen (HNA) (Mouse, MAB1281, 1:1000; Millipore), anti-type I collagen (COL1) (Rabbit, ab34710, 1:200; Abcam, Cambridge, UK), anti-type II collagen (COL2) (Mouse, MA5-12789, 1:100; Invitrogen), and anti-SOX9 (Rabbit, AB5535, 1:5000; Millipore) antibodies after blocking with Blocking One (Nacalai Tesque) for 1 h. Sections were incubated with fluorophore-labeled secondary antibodies and DAPI for 1 h at room temperature.

Techniques: Immunofluorescence, Staining

Journal: Cell Transplantation

Article Title: Laryngeal Cartilage Regeneration of Nude Rats by Transplantation of Mesenchymal Stem Cells Derived from Human-Induced Pluripotent Stem Cells

doi: 10.1177/09636897231178460

Figure Lengend Snippet: Histological findings of all rats showing regenerated cartilage-like tissue at 4 (rat A-E) and 8 (rat F-H) weeks postoperatively. The area between the yellow arrowheads indicates the defect area in the thyroid cartilage. The third column (safranin-O staining) indicates a higher magnification of the yellow box in the second column. The fourth and fifth columns indicate immunofluorescence staining, using anti-HNA, anti-SOX9, and anti-COL2 in regenerated tissue. The fourth and fifth columns indicate a higher magnification of the yellow dot box in the third column. With few cartilage-like regenerations in A and D, we indicate the cells positive for safranin-O, double-positive for HNA and SOX9, and double-positive for HNA and COL2 by the white arrow in the third, fourth, and fifth columns, respectively.

Article Snippet: Sections were incubated overnight at 4°C with anti-human nuclear antigen (HNA) (Mouse, MAB1281, 1:1000; Millipore), anti-type I collagen (COL1) (Rabbit, ab34710, 1:200; Abcam, Cambridge, UK), anti-type II collagen (COL2) (Mouse, MA5-12789, 1:100; Invitrogen), and anti-SOX9 (Rabbit, AB5535, 1:5000; Millipore) antibodies after blocking with Blocking One (Nacalai Tesque) for 1 h. Sections were incubated with fluorophore-labeled secondary antibodies and DAPI for 1 h at room temperature.

Techniques: Staining, Immunofluorescence