Journal: International Journal of Molecular Sciences

Article Title: Intraphagosomal Free Ca 2+ Changes during Phagocytosis

doi: 10.3390/ijms25084254

Figure Lengend Snippet: Sites of Ca 2+ influx marked by YFP-tagged C2 domain of PKC-γ. ( a ) A sequence of images of a RAW 264.7 cell expressing YFP–C2-γ. The phagocytic target is indicated by the crosshatched circle. At time zero (as indicated), a phagocytic cup formed at the base of the zymosan without translocation of YFP–C2-γ from the cytosol. After 1 s, translocation of YFP–C2-γ from the cytosol to the plasma membrane of the phagocytic cup is seen. This is also shown in the enlarged image ( b ). At 2 s, translocation of YFP–C2-γ to the phagocytic cup and the rest of the plasma membrane is obvious, as can also be seen in the image marked “3 s”. ( b ) Enlarged images showing a previous old phagosome marked by an “X”, which has no translocated YFP–C2-γ on its membrane at 1 s, but in the lower image at 2 s, some translation had occurred. In image ( c ) at 20 s, translocation of YFP–C2-γ had remained globally on the plasma membrane and can also be seen more clearly on the previously internalised phagosome. ( d ) The time course of the relative intensity changes measured at time t (It) as a fraction of the intensity at time zero (I 0 ), measured within the measurements zones indicated at the plasma membrane (PM) and cytosol (within the box) indicated in ( e ).

Article Snippet: RAW 264.7 cells were electroporated to introduce the C2-γ-YFP plasmid (3 µg plasmid DNA per 2 × 10 6 cells) using the Cell Line Nucleofector (Lonza) as described previously [ ].

Techniques: Sequencing, Expressing, Translocation Assay, Membrane

Journal: Oncology Letters

Article Title: Construction of recombinant pEGFP-N1-hPer2 plasmid and its expression in osteosarcoma cells

doi: 10.3892/ol.2016.4291

Figure Lengend Snippet: Schematic representation of the construction of pEGFP-N1-hPer2 plasmid.

Article Snippet: The MG63 osteosarcoma cell line was purchased from the Cell Bank of Wuhan University (Wuhan, China); the pMD19-T vector, Escherichia coli DH5a and DNA marker were purchased from Transgen Biotechnology (Beijing, China); Pst I and Kpn I restriction enzymes were purchased from NEB (Ipswich, MA, USA); the plasmid pEGFP-N1 was purchased from Clonetech Biotechnology (Mountain View, CA, USA); the DNA ligation kit, RevertAidTM First Strand cDNA synthesis kit, dNTPs, RevertAid reverse transcriptase and HiFi DNA polymerase were purchased from Fermentas (Beijing, China); TRIzolTM reagent, Lipofectamine 2000 and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Beyotime Institute of Biotechnology (Shanghai, China); AxyPrep DNA gel extraction kit was purchased from Takara Biotechnology, Inc. (Dalian, China); rabbit anti-hPer2 polyclonal antibody was purchased from ProteinTech Group, Inc. (Chicago, IL, USA; catalog no., 20359–1-AP); and Western Lighting Plus Chemiluminescence was purchased from PerkinElmer, Inc. (Waltham, MA, USA).

Techniques: Plasmid Preparation

Journal: Oncology Letters

Article Title: Construction of recombinant pEGFP-N1-hPer2 plasmid and its expression in osteosarcoma cells

doi: 10.3892/ol.2016.4291

Figure Lengend Snippet: Sequencing results of pEGFP-N1-hPer2 plasmid (partial).

Article Snippet: The MG63 osteosarcoma cell line was purchased from the Cell Bank of Wuhan University (Wuhan, China); the pMD19-T vector, Escherichia coli DH5a and DNA marker were purchased from Transgen Biotechnology (Beijing, China); Pst I and Kpn I restriction enzymes were purchased from NEB (Ipswich, MA, USA); the plasmid pEGFP-N1 was purchased from Clonetech Biotechnology (Mountain View, CA, USA); the DNA ligation kit, RevertAidTM First Strand cDNA synthesis kit, dNTPs, RevertAid reverse transcriptase and HiFi DNA polymerase were purchased from Fermentas (Beijing, China); TRIzolTM reagent, Lipofectamine 2000 and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Beyotime Institute of Biotechnology (Shanghai, China); AxyPrep DNA gel extraction kit was purchased from Takara Biotechnology, Inc. (Dalian, China); rabbit anti-hPer2 polyclonal antibody was purchased from ProteinTech Group, Inc. (Chicago, IL, USA; catalog no., 20359–1-AP); and Western Lighting Plus Chemiluminescence was purchased from PerkinElmer, Inc. (Waltham, MA, USA).

Techniques: Sequencing, Plasmid Preparation

Journal: Oncology Letters

Article Title: Construction of recombinant pEGFP-N1-hPer2 plasmid and its expression in osteosarcoma cells

doi: 10.3892/ol.2016.4291

Figure Lengend Snippet: hPer2 cDNA amplified by reverse transcription-polymerase chain reaction. M, DL8000 marker; 1, hPer2 PCR products (3765 bp).

Article Snippet: The MG63 osteosarcoma cell line was purchased from the Cell Bank of Wuhan University (Wuhan, China); the pMD19-T vector, Escherichia coli DH5a and DNA marker were purchased from Transgen Biotechnology (Beijing, China); Pst I and Kpn I restriction enzymes were purchased from NEB (Ipswich, MA, USA); the plasmid pEGFP-N1 was purchased from Clonetech Biotechnology (Mountain View, CA, USA); the DNA ligation kit, RevertAidTM First Strand cDNA synthesis kit, dNTPs, RevertAid reverse transcriptase and HiFi DNA polymerase were purchased from Fermentas (Beijing, China); TRIzolTM reagent, Lipofectamine 2000 and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Beyotime Institute of Biotechnology (Shanghai, China); AxyPrep DNA gel extraction kit was purchased from Takara Biotechnology, Inc. (Dalian, China); rabbit anti-hPer2 polyclonal antibody was purchased from ProteinTech Group, Inc. (Chicago, IL, USA; catalog no., 20359–1-AP); and Western Lighting Plus Chemiluminescence was purchased from PerkinElmer, Inc. (Waltham, MA, USA).

Techniques: Amplification, Reverse Transcription, Polymerase Chain Reaction, Marker

Journal: Oncology Letters

Article Title: Construction of recombinant pEGFP-N1-hPer2 plasmid and its expression in osteosarcoma cells

doi: 10.3892/ol.2016.4291

Figure Lengend Snippet: Identification of recombinant pEGFP-N1-hPer2 plasmid double and single enzyme digestion. M, DL8000 marker; 1, pEGFP-N1-hPer2 plasmid double digested with Pst I and Kpn I enzyme; 2, pEGFP-N1-hPer2 plasmid single digested with Pst I enzyme; 3, pEGFP-N1 plasmid.

Article Snippet: The MG63 osteosarcoma cell line was purchased from the Cell Bank of Wuhan University (Wuhan, China); the pMD19-T vector, Escherichia coli DH5a and DNA marker were purchased from Transgen Biotechnology (Beijing, China); Pst I and Kpn I restriction enzymes were purchased from NEB (Ipswich, MA, USA); the plasmid pEGFP-N1 was purchased from Clonetech Biotechnology (Mountain View, CA, USA); the DNA ligation kit, RevertAidTM First Strand cDNA synthesis kit, dNTPs, RevertAid reverse transcriptase and HiFi DNA polymerase were purchased from Fermentas (Beijing, China); TRIzolTM reagent, Lipofectamine 2000 and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Beyotime Institute of Biotechnology (Shanghai, China); AxyPrep DNA gel extraction kit was purchased from Takara Biotechnology, Inc. (Dalian, China); rabbit anti-hPer2 polyclonal antibody was purchased from ProteinTech Group, Inc. (Chicago, IL, USA; catalog no., 20359–1-AP); and Western Lighting Plus Chemiluminescence was purchased from PerkinElmer, Inc. (Waltham, MA, USA).

Techniques: Recombinant, Plasmid Preparation, Marker

Journal: Oncology Letters

Article Title: Construction of recombinant pEGFP-N1-hPer2 plasmid and its expression in osteosarcoma cells

doi: 10.3892/ol.2016.4291

Figure Lengend Snippet: Green fluorescence protein expression in MG63 cells observed by fluorescence microscope (magnification, x100). (A) pEGFP-N1 group; (B) pEGFP-N1-hPer2 group.

Article Snippet: The MG63 osteosarcoma cell line was purchased from the Cell Bank of Wuhan University (Wuhan, China); the pMD19-T vector, Escherichia coli DH5a and DNA marker were purchased from Transgen Biotechnology (Beijing, China); Pst I and Kpn I restriction enzymes were purchased from NEB (Ipswich, MA, USA); the plasmid pEGFP-N1 was purchased from Clonetech Biotechnology (Mountain View, CA, USA); the DNA ligation kit, RevertAidTM First Strand cDNA synthesis kit, dNTPs, RevertAid reverse transcriptase and HiFi DNA polymerase were purchased from Fermentas (Beijing, China); TRIzolTM reagent, Lipofectamine 2000 and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Beyotime Institute of Biotechnology (Shanghai, China); AxyPrep DNA gel extraction kit was purchased from Takara Biotechnology, Inc. (Dalian, China); rabbit anti-hPer2 polyclonal antibody was purchased from ProteinTech Group, Inc. (Chicago, IL, USA; catalog no., 20359–1-AP); and Western Lighting Plus Chemiluminescence was purchased from PerkinElmer, Inc. (Waltham, MA, USA).

Techniques: Fluorescence, Expressing, Microscopy

Journal: Oncology Letters

Article Title: Construction of recombinant pEGFP-N1-hPer2 plasmid and its expression in osteosarcoma cells

doi: 10.3892/ol.2016.4291

Figure Lengend Snippet: Expression of hPer2 mRNA and hPer2 protein. hPer2, pEGFP-N1-hPer2 group; Blank, pEGFP-N1 group; NC, control group. *P<0.05.

Article Snippet: The MG63 osteosarcoma cell line was purchased from the Cell Bank of Wuhan University (Wuhan, China); the pMD19-T vector, Escherichia coli DH5a and DNA marker were purchased from Transgen Biotechnology (Beijing, China); Pst I and Kpn I restriction enzymes were purchased from NEB (Ipswich, MA, USA); the plasmid pEGFP-N1 was purchased from Clonetech Biotechnology (Mountain View, CA, USA); the DNA ligation kit, RevertAidTM First Strand cDNA synthesis kit, dNTPs, RevertAid reverse transcriptase and HiFi DNA polymerase were purchased from Fermentas (Beijing, China); TRIzolTM reagent, Lipofectamine 2000 and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Beyotime Institute of Biotechnology (Shanghai, China); AxyPrep DNA gel extraction kit was purchased from Takara Biotechnology, Inc. (Dalian, China); rabbit anti-hPer2 polyclonal antibody was purchased from ProteinTech Group, Inc. (Chicago, IL, USA; catalog no., 20359–1-AP); and Western Lighting Plus Chemiluminescence was purchased from PerkinElmer, Inc. (Waltham, MA, USA).

Techniques: Expressing, Control