Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Reg1cp is involved in the regulation of CD31 hi EMCN hi endothelium formation. (A) Reg1cp expression levels in different cells isolated from human bone marrow. (B) Reg1cp expression levels in ECs. (C) The predicted secondary structure of Reg1cp and mutant Reg1cp around the mutant site (RNAfold Webserver, http://rna.tbi.univie.ac.at/cgibin/RNAfold.cgi ). (D) FACS analysis dot plot and quantitation of CD31 hi EMCN hi ECs (Type H ECs) of bone samples from one 37-yr-old male Reg1cp +/mut subject and five age-matched Reg1cp +/+ controls. (E) qRT-PCR analysis of CD31 and EMCN expression levels in ECs. (F) qRT-PCR analysis of VEGFA, VEGFB , PDGFA , PDFB , TGFβ , and FGF1 expression level in ECs. (G) Reg1cp expression level in human ECs transfected with Reg1cp-mut or Reg1cp-wt plasmid. (H and I) CD31 and EMCN expression levels (H) and VEGFA, VEGFB , PDGFA , PDFB , TGFβ , and FGF1 expression levels (I) in ECs. (J–L) Representative images of Alizarin Red S staining (J) and qRT-PCR analysis of the levels of SP7 and RUNX2 expression (K and L) in human BMSCs transfected with Reg1cp-mut or Reg1cp-wt plasmid with osteogenic induction. Scale bar, 0.5 cm. In A, B, E–I, K, and L, n = 5 in each group from three independent experiments. J is representative of three independent experiments. Data are shown as the mean ± SD. *, P < 0.05; **, P < 0.01; N.S, no significance; Student’s t test (B, E, and F) and ANOVA (A, G–I, K, and L). BMEPC, bone marrow endothelial progenitor cell; OB, osteoblast; PRE-OC, osteoclast precursor cell.

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Expressing, Isolation, Mutagenesis, Quantitation Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, Staining

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Mutant Reg1cp abolishes the role of KLF3 in CD31 hi EMCN hi vessel formation. (A) ChIP-PCR assays with anti-KLF3 antibodies or anti-IgG antibodies using specific primers targeting the promoter regions of JUNB . (B) qRT-PCR analysis of the JUNB expression level after anti-KLF3 or anti-IgG ChIP. (C) HMECs were transfected with luciferase reporter carrying WT-pGL3-JunB or MUT-pGL3-JunB, respectively, and cotransfected with the Klf3 plasmid or vector. Firefly luciferase values, normalized for renilla luciferase, are presented. (D and E) Western blotting analysis (D) and quantitation (E) of the relative levels of KLF3, JUNB, and VEGFA protein expression. (F) ChIP-PCR assays with anti-KLF3 antibodies or anti-IgG antibodies in HMECs transfected with Reg1cp-mut or Reg1cp-wt plasmids. (G) JUNB expression level of anti-KLF3 or anti-IgG ChIP. (H and I) Western blotting analysis (H) and quantitation (I) of the relative levels of KLF3, JUNB, and VEGFA protein expression. (J) ChIP-PCR assays with anti-KLF3 antibodies or anti-IgG antibodies in HMECs with different Reg1cp genotypes. Het , heterozygous mutation; Hom , homozygous mutation. (K) JUNB expression level of anti-KLF3 or anti-IgG ChIP. (L and M) Western blotting analysis (L) and quantitation (M) of the relative levels of JUNB and VEGFA protein expression. (N and O) Representative images (N) and relative quantification (O) of a transwell migration assay. Scale bar, 150 µm. (P and Q) Representative images (P) and relative quantification (Q) of tube branch numbers of a Matrigel tube formation assay. Scale bar, 750 µm. (R and S) HMECs were cultured under hypoxia for 24 h and analyzed (R) and quantified (S) for VEGFR2 phosphorylation (pVEGFR2, top) and VEGFR2 total levels (bottom). All panels were representative of three independent experiments. Data are shown as the mean ± SD. **, P < 0.01; Student’s t test (B, C, E, G, I, and K) and ANOVA (M, O, Q, and S).

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Mutagenesis, Quantitative RT-PCR, Expressing, Transfection, Luciferase, Plasmid Preparation, Western Blot, Quantitation Assay, Quantitative Proteomics, Transwell Migration Assay, Tube Formation Assay, Cell Culture, Phospho-proteomics

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Endothelial-specific Klf3 knockout mice show increased CD31 hi EMCN hi vessels and bone formation. (A) Expression level of Klf3 in ECs. (B) qRT-PCR analysis of JunB and Vegfa levels in ECs. (C and D) Representative images (C) and quantitation (D) of CD31 (green) and EMCN (red) immunostaining in femora from endothelial-specific Klf3 knockout mice ( Klf3 cdh5 −/− ) and their littermate controls ( Klf3 flox/flox ). Scale bars, 100 µm. (E and F) Quantitation (E) and FACS analysis dot plot (F) of CD31 hi EMCN hi ECs (Type H ECs) from long bones of 1-, 3-, and 12-mo-old Klf3 cdh5 −/− mice and their littermate controls. (G–K) Representative μCT images (G) and quantitative μCT analysis (H–K) of trabecular bone microarchitecture in femora. (L and M) Immunohistochemical staining (L) and quantification (M) of OCN + cells (brown) in femora. Scale bar, 50 µm. (N–P) Representative images of calcein double labeling of trabecular bone (N) with quantification of BFR per bone surface (BFR/BS; O) and MAR (P). Scale bar, 25 µm. (Q and R) Serum levels of OCN (Q) and CTX (R) at the time of harvest. n = 6 mice in each group from three independent experiments. Data are shown as the mean ± SD. *, P < 0.05; **, P < 0.01. Student’s t test.

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Knock-Out, Expressing, Quantitative RT-PCR, Quantitation Assay, Immunostaining, Immunohistochemical staining, Staining, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Endothelial-specific Klf3 knockout in OVX mice show increased CD31 hi EMCN hi vessels and bone formation. (A and B) FACS analysis dot plot (A) and quantification (B) of CD31 hi EMCN hi ECs (Type H ECs). (C and D) Representative μCT images (C) and quantitative μCT analysis (D) of trabecular bone microarchitecture in femora. (E and F) Serum levels of OCN ( E) and CTX (F) at the time of harvest. (G and H) Representative images (G) and quantification (H) of ALP (green) immunostaining in femora. Scale bar, 200 µm. (I and J) Immunohistochemical staining (I) and quantification (J) of OCN + cells (brown) in femora. Scale bar, 50 µm. (K and L) Immunohistochemical staining (K) and quantification (L) of COL 1 (green) in femora. Scale bar, 200 µm. (M) Representative images of TRAP staining of femora. Scale bar, 50 µm. (N) Quantification data of TRAP + cells in trabecular bone surface. Number of TRAP + cells per bone perimeter (Tb.N.Trap + /B.Pm) was measured. n = 6 mice in each group from three independent experiments. Data are shown as the mean ± SD. **, P < 0.01; ANOVA.

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Knock-Out, Immunostaining, Immunohistochemical staining, Staining

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: A natural compound is identified as a KLF3 inhibitor by molecular docking. (A–D) qRT-PCR analysis of the relative levels of CD31 (A), EMCN (B), JUNB (C), and VEGFA (D) expression in HMECs treated with four different compounds. n = 3 in each group from three independent experiments. (E) The structure of Ophiopogonin D selected by molecular docking. (F) Crystal structure of Ophiopogonin D bound to Klf3. (G) HPLC-MS chromatograms of Ophiopogonin D reference substance (upper panel) and KLF3 recruit ligand (lower panel). Representative of two independent experiments. (H) ChIP-PCR assays with anti-Klf3 antibodies or anti-IgG antibodies in HMECs treated with Ophiopogonin D and control groups. Representative of three independent experiments. (I) qRT-PCR analysis of JUNB expression after anti-KLF3 or anti-IgG ChIP. n = 3 in each group from three independent experiments. (J and K) Western blotting analysis (J) and the quantification (K) of the levels of JUNB and VEGFA in HMECs treated with vehicle or different doses of Ophiopogonin D. Representative of three independent experiments. Data are shown as the mean ± SD. *, P < 0.05; **, P < 0.01; Student’s t test (A–D and I) and ANOVA (K).

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Ophiopogonin D treatment promotes CD31 hi EMCN hi vessels and bone formation in aged mice. 12-mo-old C57/B6 mice were intraperitoneally treated with Ophiopogonin D at 20 mg/kg every other day for 3 mo. (A) qRT-PCR analysis of JunB level in ECs. (B) qRT-PCR analysis of Vegfa level in ECs. (C and D) FACS analysis dot plot (C) and quantification (D) of CD31 hi Emcn hi ECs (Type H ECs). (E and F) Representative images (E) and quantification (F) of CD31 (green) and EMCN (red) immunostaining in femora from Ophiopogonin D–treated mice and the vehicle control group. G, growth plate. B, bone. Scale bar, 100 µm. (G–K) Representative μCT images (G) and quantitative μCT analysis (H–K) of trabecular bone microarchitecture in femora. (L and M) Representative images (L) and quantification (M) of ALP (green) immunostaining in femora. Scale bar, 200 µm. (N and O) Immunohistochemical staining (N) and quantification (O) of OCN + cells (brown) in femora. Scale bar, 50 µm. (P and Q) Immunohistochemical staining (P) and quantification (Q) of COL 1 (green) in femora. Scale bar, 200 µm. (R and S) Serum levels of OCN (R) and CTX (S) at the time of harvest. (T–V) Representative images of calcein double labeling of trabecular bone (T) with quantification of BFR/BS (U) and MAR (V). Scale bar, 25 µm. n = 6 mice in each group from three independent experiments. Data are shown as the mean ± SD. *, P < 0.05; **, P < 0.01; ANOVA. Tb. BV/TV, trabecular bone volume per tissue volume; Tb. N, trabecular number; Tb. Sp, trabecular separation; Tb. Th, trabecular thickness.

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Quantitative RT-PCR, Immunostaining, Control, Immunohistochemical staining, Staining, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Ophiopogonin D treatment promotes CD31 hi EMCN hi vessels and bone formation in OVX mice. 2-mo-old C57/B6 mice underwent OVX surgery and were intraperitoneally treated with Ophiopogonin D at 20 mg/kg every other day for 3 mo. (A and B) FACS analysis dot plot (A) and quantification (B) of CD31 hi EMCN hi ECs (Type H ECs). (C–G) Representative μCT images (C) and quantitative μCT analysis (D–G) of trabecular bone microarchitecture in femora. (H and I) Serum levels of OCN (H) and CTX (I) at the time of harvest. (J and K) Representative images (J) and quantification (K) of ALP (green) immunostaining in femora. Scale bar, 200 µm. (L and M) Immunohistochemical staining (L) and quantification (M) of COL 1 (green) in femora. Scale bar, 200 µm. (N and O) Immunohistochemical staining (N) and quantification (O) of OCN + cells (brown) in femora. Scale bar, 50 µm. (P) Representative images of TRAP staining of femora from Ophiopogonin D–treated mice and their controls. (Q) Quantification of TRAP + cells in trabecular bone surfaces. Number of TRAP + cells per bone perimeter (Tb.N.Trap + /B.Pm) was measured. Scale bar, 50 µm. ( n = 6 mice in each group from three independent experiments. Data are shown as the mean ± SD. **, P < 0.01; ANOVA (B and D–G) and Student’s t test (H, I, K, M, O, and Q). Tb. BV/TV, trabecular bone volume per tissue volume; Tb. N, trabecular number; Tb. Sp, trabecular separation; Tb Th, trabecular thickness.

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Immunostaining, Immunohistochemical staining, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Conditions Mimicking the Cancer Microenvironment Modulate the Functional Outcome of Human Chorionic Villus Mesenchymal Stem/Stromal Cells in vitro

doi: 10.3389/fcell.2021.650125

Figure Lengend Snippet: Effect of CM-MDA231 on CV-MSCs expression of Adhesion molecules. Flow cytometry analysis of the adhesion molecules performed on preconditioned, in-treatment, and untreated CV-MSCs showed that there was significant increase in the expression levels for VCAM, ICAM1 and PECAM, after preconditioning them for 72 h (A–C) , but no changes in the expression levels were observed in cells preconditioned for 24 h, in-treatment and untreated controls. No significant changes in expression levels was observed for E-Cadherin (D) between the preconditioned, in-treatment and untreated controls (Panel 1). The data obtained by FACS from five independent experiments was quantified. The average is presented as bar diagrams in panel 2. VCAM is represented as (A) , ICAM1 as (B) , PECAM as (C) and E-Cadherin as (D) in panel 2, respectively. Bars represent standard errors * P ≤ 0.05.

Article Snippet: GAPDH and a panel of fluorescent-labeled antibodies (VCAM (cat#FAB5649P), ICAM (cat#BBA20), PECAM (cat#FAB3567P), E-Cadherin (cat#FAB18381P), Integrin α5 (cat#FAB1864P), Integrin-M (cat#FAB16991P), and EpCAM (cat#FAB9601P), used for flow cytometry were purchased from R&D Systems, MN, United States. p53 (cat#2527), pRb (S807) (cat#8516), pChk2 (T68) (cat#2661) and β-Actin (cat#3700 and cat#8457) antibodies for immunoblotting were purchased from Cell Signaling Technologies, MA, United States.

Techniques: Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: A novel strategy to engineer pre-vascularized 3-dimensional skin substitutes to achieve efficient, functional engraftment

doi: 10.1038/s41598-019-44113-6

Figure Lengend Snippet: Interconnected vascular network formation in 3D skin substitutes 7 days after epidermal differentiation. ( a – c ) HUVEC were seeded at 0.1 to 1 × 10 5 cells per culture insert; FN-G-coated NHDF content (1 × 10 7 cells) remained constant. FN-G-coated NHDF and HUVEC were cocultured at 1,000:1, 500:1, or 100:1, and then with HEKn (1 × 10 6 cells). ( a ) Whole-mount CD31 immunofluorescence analysis revealed that HUVEC developed branching vessels within the dermal layer. The data are representative of 2 independent experiments (n = 6/condition). ( b , c ) Quantification of vascular structures to determine the vessel density ( b ) and branching index (branching points/unit area) ( c ). The data are the mean ± SD (n = 6). * p < 0.05 and ** p < 0.01 determined by one-way ANOVA with Tukey’s multiple comparison post hoc test.

Article Snippet: The following primary antibodies were used for immunostaining of: a human–specific CD31 (1:200, NBP2-15202, clone C31.3; Novus Biologicals, Centennial, CO, USA), a mouse–specific CD31 (1:100, 14-0311-82, clone 390; Invitrogen), HLA-Class I ABC (1:2,500, ab70328, clone: EMR8-5; Abcam), and laminin 5 (1:200; ab14509, Abcam).

Techniques: Immunofluorescence, Comparison

Journal: Scientific Reports

Article Title: A novel strategy to engineer pre-vascularized 3-dimensional skin substitutes to achieve efficient, functional engraftment

doi: 10.1038/s41598-019-44113-6

Figure Lengend Snippet: In vitro evaluation of the therapeutic potential of pre-vascularized 3D skin substitutes. ( a – e ) HUVEC (0.2 × 10 5 cells/insert) mixed with FN-G-coated NHDF were cocultured, subsequently covered with HEKn, then cultured for up to an additional 7 days. ( a ) Macroscopic view of the construct in the culture insert. Scale bar: 10 mm. ( b – e ) Histological and immunohistochemical staining with hematoxylin and eosin ( b ), Masson’s trichrome ( c ), anti-laminin 5 ( d , arrows = basement membrane), and anti-CD31 (e, arrows = CD31 + blood vessel). Scale bars: 500 μm ( b ), 100 μm ( c – e ).

Article Snippet: The following primary antibodies were used for immunostaining of: a human–specific CD31 (1:200, NBP2-15202, clone C31.3; Novus Biologicals, Centennial, CO, USA), a mouse–specific CD31 (1:100, 14-0311-82, clone 390; Invitrogen), HLA-Class I ABC (1:2,500, ab70328, clone: EMR8-5; Abcam), and laminin 5 (1:200; ab14509, Abcam).

Techniques: In Vitro, Cell Culture, Construct, Immunohistochemical staining, Staining, Membrane

Journal: Scientific Reports

Article Title: A novel strategy to engineer pre-vascularized 3-dimensional skin substitutes to achieve efficient, functional engraftment

doi: 10.1038/s41598-019-44113-6

Figure Lengend Snippet: Quantification of wound vasculature in vivo . ( a ) Immunohistochemical detection of mouse-specific CD31 (mCD31) and human-specific CD31 (hCD31) at 7 and 14 days after grafting. Dashed lines indicate the skin substitute–host interface. Scale bars: 100 μm. ( b ) Quantification of CD31 + blood vessels in the dermal areas of grafts. The data are the mean ± SD (n = 5). ** p < 0.01 compared with the non-vascularized control (unpaired Student’s t -test). ( c ) Immunofluorescence image stained for mouse-CD31 (red) and human-CD31 (green) of pre-vascularized substitute-treated wounds at day 14 after grafting. Nuclei were stained with DAPI (blue). White arrowheads indicate human–mouse chimeric vessel. Scale bars: 50 μm.

Article Snippet: The following primary antibodies were used for immunostaining of: a human–specific CD31 (1:200, NBP2-15202, clone C31.3; Novus Biologicals, Centennial, CO, USA), a mouse–specific CD31 (1:100, 14-0311-82, clone 390; Invitrogen), HLA-Class I ABC (1:2,500, ab70328, clone: EMR8-5; Abcam), and laminin 5 (1:200; ab14509, Abcam).

Techniques: In Vivo, Immunohistochemical staining, Control, Immunofluorescence, Staining