Journal: Journal of Cellular Physiology

Article Title: Interleukin‐37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction

doi: 10.1002/jcp.27526

Figure Lengend Snippet: IL‐37b suppressed RANKL‐induced osteoclast differentiation without inducing cytotoxicity. (a) RAW264.7 cells were seeded onto 96‐well plates and incubated for 24 or 72 hr with different concentrations of IL‐37b. The CCK‐8 assay was used to detect cell proliferation. (b) Precursor cells were cultured with various concentrations of IL‐37b followed by M‐CSF (50 ng/ml) and RANKL (50 ng/ml) stimulation for 72 hr. These cells were simultaneously exposed to all of these factors. The steps for TRAP staining are described in the methods. Scale bar = 200 μm. (c) TRAP‐positive multinucleated cells ( ≥ 3 nuclei) were identified as osteoclasts and were counted. (d) BMMs were treated with M‐CSF (50 ng/ml), RANKL (50 ng/ml), and IL‐37 (200 ng/ml) for 5 days. (e) RAW264.7 cells were plated on the Osteo Assay Surface and were cultured with RANKL and M‐CSF for 6 days in the presence or absence of 200 ng/ml IL‐37b. Scale bar = 200 μm. Quantification of the bone resorption area on the Osteo Assay Surface. N = 4. The data are presented as the means ± SD. * p < 0.05, ** p < 0.01. CCK‐8: cell counting kit‐8; IL‐37b: interleukin‐37b; M‐CSF: macrophage colony stimulating factor; RANKL: receptor activator of nuclear factor‐κB ligand; TRAP: tartrate‐resistant acid phosphatase [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Recombinant human IL‐37b/IL‐1F7b protein (7585‐IL‐025/CF), recombinant murine RANKL (462‐TEC‐010/CF), and macrophage colony stimulating factor (M‐CSF; 416‐ML‐010) were from R&D Systems (Minneapolis, MN).

Techniques: Incubation, CCK-8 Assay, Cell Culture, Staining, Cell Counting

Journal: Journal of Cellular Physiology

Article Title: Interleukin‐37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction

doi: 10.1002/jcp.27526

Figure Lengend Snippet: IL‐37b inhibited the expression of specific mRNAs and proteins at different stages of osteoclast differentiation. The MyD88 dimerization inhibitor (ST 2825) reversed the effects of IL‐37b on osteoclastogenesis. (a) Cells were treated with RANKL (50 ng/ml), M‐CSF (50 ng/ml), and IL‐37b (200 ng/ml) for 24 or 72 hr, and then lysed for Western blot analyses using antibodies against NFATc1 and GAPDH. (b,c) The expression of genes associated with osteoclast differentiation, fusion, and function were detected by qRT‐PCR. * p < 0.05 and ** p < 0.01 compared with the RANKL‐treated control. (d) TRAP staining showed that 10 μg/ml ST 2825 partially counteracted the inhibitory effects of IL‐37b on osteoclast formation. Scale bar = 100 μm. (e) Western blot showing the level of NFATc1 in cells stimulated with RANKL in the presence or absence of ST 2825. The results were normalized to GAPDH levels. (f) Co‐IP analysis of the interaction between SIGIRR and MyD88 in RAW264.7 cells treated with RANKL and IL‐37b. Co‐IP: coimmunoprecipitation; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; IL‐37b: Interleukin‐37b; mRNA: messenger RNA; MyD88: myeloid differentiation factor 88; M‐CSF: macrophage colony stimulating factor; NFATc1: nuclear factor of activated T cells 1; qRT‐PCR: quantitative real‐time polymerase chain reaction; RANKL: receptor activator of nuclear factor‐κB ligand; SIGIRR: single Ig IL‐1‐related receptor; TRAP: tartrate‐resistant acid phosphatase [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Recombinant human IL‐37b/IL‐1F7b protein (7585‐IL‐025/CF), recombinant murine RANKL (462‐TEC‐010/CF), and macrophage colony stimulating factor (M‐CSF; 416‐ML‐010) were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Staining, Co-Immunoprecipitation Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Cellular Physiology

Article Title: Interleukin‐37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction

doi: 10.1002/jcp.27526

Figure Lengend Snippet: IL‐37b decreased the phosphorylation of p65 and IκB, intermediates in the classical NF‐κB pathway, in cells stimulated with RANKL, thereby reducing the translocation of the NF‐κB p65 subunit into the nucleus. (a) RAW264.7 cells were plated in six‐well plates and pretreated with or without IL‐37b (200 ng/ml) for 2 hr in the presence of M‐CSF (50 ng/ml) before RANKL stimulation for 0, 5, 15, and 30 min. Whole‐cell lysates were subjected to Western blot analyses with the indicated antibodies. GAPDH was used as the internal control. (b) RAW264.7 cells were seeded in 96‐well plates and treated with IL‐37b (200 ng/ml) for 2 hr, followed by stimulation with 50 ng/mll RANKL for 30 min. The intracellular location of the p65 subunit was observed by immunofluorescence staining. Five cells were selected from the RANKL group and the IL‐37b intervention group, respectively. The gray values of the red and blue staining were measured using the ImageJ software, and the mean values were plotted using excel. Scale bar = 200 μm. DAPI: 4’,6‐diamidino‐2‐phenylindole; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; IL‐37b: interleukin‐37b; M‐CSF: macrophage colony stimulating factor; NF‐κB: nuclear factor‐κB; RANKL: receptor activator of nuclear factor‐κB ligand [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Recombinant human IL‐37b/IL‐1F7b protein (7585‐IL‐025/CF), recombinant murine RANKL (462‐TEC‐010/CF), and macrophage colony stimulating factor (M‐CSF; 416‐ML‐010) were from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Control, Immunofluorescence, Staining, Software

Journal: Journal of Cellular Physiology

Article Title: Interleukin‐37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction

doi: 10.1002/jcp.27526

Figure Lengend Snippet: RAW264.7 cells that had been pretreated with RANKL for 24 hr were stimulated with LPS (100 ng/ml) to induce their differentiation into osteoclasts; IL‐37b reversed the process. (a) The CCK‐8 assay was used to examine the effect of LPS on progenitor cell proliferation. (b) IL‐37b inhibits RANKL or LPS‐mediated osteoclast fusion. A 24‐hr RANKL pretreatment triggered the fusion of precursor cells exposed to LPS conditions. Immunofluorescence staining revealed that IL‐37b reduced LPS‐induced actin ring formation. Scale bar = 200 μm. (c) After culture under the same induction conditions described above for three days, TRAP‐positive multinucleated cells were identified as osteoclasts. Scale bar = 200 μm. (d) RAW264.7 cells were treated with RANKL (50 ng/ml) for 24 hr and then stimulated with LPS (100 ng/ml) or LPS (100 ng/ml) and IL‐37b (200 ng/ml) for 24 hr. M‐CSF (50 ng/ml) was maintained in the culture media throughout the induction period. The relative expression levels of NFATc1 and c‐Fos were detected by quantitative PCR. (e) IL‐37b reduces the expression of proinflammatory cytokines during osteoclast differentiation. The levels of the IL‐1β, IL‐6, and TNF‐α mRNAs during the course of RANKL‐ or LPS‐induced osteoclastogenesis were analyzed by qRT‐PCR. *p < 0.05; ** p < 0.01 compared with the LPS‐treated group. CCK‐8: cell counting kit‐8; IL‐37b: interleukin‐37b; LPS: lipopolysaccharide; M‐CSF: macrophage colony stimulating factor; NFATc1: nuclear factor of activated T cells 1; qRT‐PCR: quantitative real‐time polymerase chain reaction; RANKL: receptor activator of nuclear factor‐κB ligand; TNF: tumor necrosis factor; TRAP: tartrate‐resistant acid phosphatase [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Recombinant human IL‐37b/IL‐1F7b protein (7585‐IL‐025/CF), recombinant murine RANKL (462‐TEC‐010/CF), and macrophage colony stimulating factor (M‐CSF; 416‐ML‐010) were from R&D Systems (Minneapolis, MN).

Techniques: CCK-8 Assay, Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cell Counting

Journal: Clinical and Vaccine Immunology

Article Title: Comparative Measurement of Cell-Mediated Immune Responses of Swine to the M and N Proteins of Porcine Reproductive and Respiratory Syndrome Virus

doi: 10.1128/cvi.00365-09

Figure Lengend Snippet: FIG. 1. Cloning of viral genes into protein expression vectors. (A) The recombinant TAT-PRRSV-M and TAT-PRRSV-N genes were cloned into the pDrive and pCR2.1 cloning vectors, respectively. Once they were cloned into the cloning vectors, the insert DNAs were subcloned into the pQE30 and pQE30-UA E. coli expression vectors. (B) The porcine GM-CSF gene was cloned into the pcDNA3.1 mammalian protein expression vector. orf6 and orf7 of PRRSV cloned in the pCR2.1 vector were subcloned downstream of the GM-CSF gene.

Article Snippet: GM-CSF expression was identified with mouse antiporcine GM-CSF antibody (R&D Systems) and goat anti-mouse IgG (Biosource).

Techniques: Cloning, Expressing, Recombinant, Clone Assay, Plasmid Preparation

Journal: Clinical and Vaccine Immunology

Article Title: Comparative Measurement of Cell-Mediated Immune Responses of Swine to the M and N Proteins of Porcine Reproductive and Respiratory Syndrome Virus

doi: 10.1128/cvi.00365-09

Figure Lengend Snippet: FIG. 3. Generation of monocyte-derived DCs. The levels of expression of DC markers were identified in the immature DCs (A) and mature DCs (B). The immature DCs were generated from monocytes by culturing PBMCs with GM-CSF and IL-4 for 6 days. The immature DCs became mature DCs as the result of 1 day of treatment with TNF-. The levels of expression of MHC class I, MHC class II, CD172a, and CD80/CD86 molecules were determined by FACS analysis. The expression of MHC class I, MHC class II, and CD172a molecules was determined by the use of an FITC-conjugated monoclonal antibody specific for each corresponding molecule. The expression of CD80/CD86 was determined with an FITC-conjugated human CD152Ig protein. The x and y axes in each panel indicate the cell count and fluorescent intensity, respectively.

Article Snippet: GM-CSF expression was identified with mouse antiporcine GM-CSF antibody (R&D Systems) and goat anti-mouse IgG (Biosource).

Techniques: Derivative Assay, Expressing, Generated, Cell Counting

Journal: Clinical and Vaccine Immunology

Article Title: Comparative Measurement of Cell-Mediated Immune Responses of Swine to the M and N Proteins of Porcine Reproductive and Respiratory Syndrome Virus

doi: 10.1128/cvi.00365-09

Figure Lengend Snippet: FIG. 5. Identification of the recombinant genes and proteins ex- pressed in mammalian cells. The mammalian expression vectors en- coding the GM-CSF-PRRSV-M and GM-CSF-PRRSV-N proteins were transfected into 3T3 cells. After 2 days, RT-PCR and Western blotting were conducted to identify the expression of the recombinant genes and proteins in the cells. (A) RT-PCR for the detection of the recombinant GM-CSF-PRRSV-M gene. Lane 1, standard DNA marker; lane 2, cells transfected with the empty vector; lane 3, cells transfected with the pcDNA3.1-GM-CSF-PRRSV-M vector. (B) RT- PCR for the detection of the recombinant GM-CSF-PRRSV-N gene. Lane 1, standard DNA marker; lane 2, cells transfected with empty vector; lane 3, cells transfected with the pcDNA3.1-GM-CSF- PRRSV-N vector. (C) Western blotting for the detection of the re- combinant GM-CSF-PRRSV-M protein. Lane 1, standard protein marker; lane 2, cells transfected with the empty vector; lane 3, cells transfected with the pcDNA3.1-GM-CSF-PRRSV-M vector. (D) Western blotting for the detection of the recombinant GM-CSF- PRRSV-N protein. Lane 1, standard protein marker; lane 2, cells transfected with the empty vector; lane 3, cells transfected with the pcDNA3.1-GM-CSF-PRRSV-N vector.

Article Snippet: GM-CSF expression was identified with mouse antiporcine GM-CSF antibody (R&D Systems) and goat anti-mouse IgG (Biosource).

Techniques: Recombinant, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Marker, Plasmid Preparation

Journal: Clinical and Vaccine Immunology

Article Title: Comparative Measurement of Cell-Mediated Immune Responses of Swine to the M and N Proteins of Porcine Reproductive and Respiratory Syndrome Virus

doi: 10.1128/cvi.00365-09

Figure Lengend Snippet: FIG. 6. Antibody levels in the immunized pigs. Pigs were immu- nized with plasmid DNAs, and the levels antibodies to the PRRSV-M and PRRSV-N proteins were determined by ELISA. (A) Levels of antibody to the PRRSV-M protein in pigs immunized with pcDNA3.1- GM-CSF-PRRSV-M. Three pigs immunized with M protein (pigs M#1 to M#3, respectively) and two nonimmunized pigs (control pigs 1 and 2 [Cont#1 and Cont #2, respectively]) were examined. (B) Lev- els of antibody to PRRSV-N protein in pigs immunized with pcDNA3.1-GM-CSF-PRRSV-N. Three immunized pigs (N#1 to N#3, respectively) and two nonimmunized pigs (controls 1 and 2 [Cont#1 and Cont #2, respectively]) were assessed.

Article Snippet: GM-CSF expression was identified with mouse antiporcine GM-CSF antibody (R&D Systems) and goat anti-mouse IgG (Biosource).

Techniques: Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Control

Journal: Clinical and Vaccine Immunology

Article Title: Comparative Measurement of Cell-Mediated Immune Responses of Swine to the M and N Proteins of Porcine Reproductive and Respiratory Syndrome Virus

doi: 10.1128/cvi.00365-09

Figure Lengend Snippet: FIG. 7. T-cell proliferation by antigen stimulation. T cell-containing PBMCs obtained from the immunized pigs and nonimmunized pigs (control pigs C1 and C2) were cocultured with APCs for 3 days at effector cell-to-APC ratios of 5:1. PBMCs and DCs transduced with TAT-conjugated PRRSV-M or PRRSV-N protein were used as APCs. Effector T cells from pigs immunized with the pcDNA3.1-GM-CSF- PRRSV-M (pigs M1 to M3) were stimulated by PBMCs (A) and DCs (C). Effector T cells from pigs immunized with pcDNA3.1-GM-CSF- PRRSV-N (pigs N1 to N3) were stimulated by PBMCs (B) and DCs (D). T-cell proliferation was determined by the MTT assay. Statistical significance was determined by Student’s t test: *, P 0.05; ***, P 0.001.

Article Snippet: GM-CSF expression was identified with mouse antiporcine GM-CSF antibody (R&D Systems) and goat anti-mouse IgG (Biosource).

Techniques: Control, Transduction, MTT Assay

Journal: Clinical and Vaccine Immunology

Article Title: Comparative Measurement of Cell-Mediated Immune Responses of Swine to the M and N Proteins of Porcine Reproductive and Respiratory Syndrome Virus

doi: 10.1128/cvi.00365-09

Figure Lengend Snippet: FIG. 8. IFN- production from T cells. T cell-containing PBMCs were obtained from immunized pigs and nonimmunized pigs (control pigs C1 and C2). They were stimulated for 3 days by APCs at an effector cell-to-APC ratio of 5:1. PBMCs and DCs transduced with TAT-conjugated PRRSV-M or PRRSV-N proteins were used as APCs. Effector T cells from pigs immunized with the pcDNA3.1-GM-CSF-PRRSV-M (pigs M1 to M3) were stimulated by PBMCs (A) and DCs (C). Effector T cells from pigs immunized with pcDNA3.1-GM-CSF-PRRSV-N (pigs N1 to N3) were stimulated by PBMCs (B) and DCs (D). The amounts of IFN- in the cell culture supernatants were determined by ELISA. Statistical significance was determined by Student t test: **, P 0.01; ***, P 0.001.

Article Snippet: GM-CSF expression was identified with mouse antiporcine GM-CSF antibody (R&D Systems) and goat anti-mouse IgG (Biosource).

Techniques: Control, Transduction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell Proliferation

Article Title: Continuous expression of reprogramming factors induces and maintains mouse pluripotency without specific growth factors and signaling inhibitors

doi: 10.1111/cpr.13090

Figure Lengend Snippet: Mouse ESCs maintained by induced expression of OSKM upon withdrawal of 2iL. A, Schematic of pluripotency maintenance in mouse ESCs via expression of OSKM without 2iL. The Tet‐On‐OSKM/Oct4‐GFP ESC line harbored a DOX (Doxycycline)‐induced single‐copy OSKM ( Oct4 , Sox2 , Klf4 , and c‐Myc ) transgenic cassette and a GFP reporter driven by endogenous Oct4 distal promoter. The culture medium was switched from N2B27 with 2iL to N2B27 with 2 μg/mL DOX. B, Colony morphology of ESCs cultured under different conditions at selected time points. Ctrl (control), N2B27 group; 2iL, N2B27 with 2iL group; and OSKM, N2B27 with DOX group. Scale bar, 75 μm. C, The percentages of GFP‐positive cells of ESCs cultured under three different conditions at selective time points. D, Morphology of ESCs cultured under 2iL and OSKM conditions, respectively. GFP‐positive cells could be observed. Scale bar, 50 μm. E, Statistical analysis of the relative cell numbers of ESCs cultured in OSKM medium (OSKM‐ESCs) and ESCs cultured in 2iL medium (2iL‐ESCs) at indicated time points. Cell Counting Kit‐8 (CCK‐8) was used for data collecting. Data were represented as mean ± SEM. ** P < .01. F, FACS analysis of DNA content of ESCs under 2iL and OSKM conditions. Percentages of cells in G1, S, and G2/M phases were shown. G, Immunostaining for ESC markers OCT4, SOX2, NANOG, and SSEA1 of OSKM‐ESCs. DNA was stained with Hoechst 33342. Scale bars, 20 μm. H, Histological section analysis of the teratomas derived from OSKM‐ESCs showed differentiation into all three germ layers (ectoderm, mesoderm, and endoderm). Scale bar, 200 μm

Article Snippet: The primary antibodies were as follows: anti‐OCT4 (Abcam, ab19857), anti‐NANOG (Abcam, ab80892), anti‐SOX2 (Abcam, ab97959), anti‐SSEA1 (Abcam, ab16285), and anti‐TUJ1 (Biolegend, 802001).

Techniques: Expressing, Transgenic Assay, Cell Culture, Cell Counting, CCK-8 Assay, Immunostaining, Staining, Derivative Assay

Journal: Cell Proliferation

Article Title: Continuous expression of reprogramming factors induces and maintains mouse pluripotency without specific growth factors and signaling inhibitors

doi: 10.1111/cpr.13090

Figure Lengend Snippet: Induction and maintenance of pluripotency via induced OSKM expression. A, Colony morphology of reprogrammed cells in different inductive media at selected time points. Groups of 2iL and OSKM were shown. Scale bar, 75 μm. B, Colonies observed after reprogramming under inductive media of 2iL and OSKM, respectively. Cells were stained by alkaline phosphatase (AP). C, Morphology of stable cell lines of OSKM‐iPSCs at Passage 23. Scale bar, 75 μm. D, AP staining of OSKM‐iPSCs. Scale bar, 75 μm. E, Karyotype analysis of OSKM‐iPS‐4 cell line. F, Statistical graph of karyological characteristics of the two OSKM‐iPSC lines (OSKM‐iPS‐4 and OSKM‐iPS‐24) at Passage 12. G, Immunostaining for ESC markers OCT4, SOX2, NANOG, and SSEA1 of OSKM‐iPSCs. DNA was stained with Hoechst 33342. Scale bars, 20 μm. H, Histological section analysis of the teratomas derived from OSKM‐iPSCs. Ectoderm, mesoderm, and endoderm structures were shown. Scale bar, 200 μm

Article Snippet: The primary antibodies were as follows: anti‐OCT4 (Abcam, ab19857), anti‐NANOG (Abcam, ab80892), anti‐SOX2 (Abcam, ab97959), anti‐SSEA1 (Abcam, ab16285), and anti‐TUJ1 (Biolegend, 802001).

Techniques: Expressing, Staining, Stable Transfection, Immunostaining, Derivative Assay