Journal: The Journal of Biological Chemistry

Article Title: NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I *

doi: 10.1074/jbc.M116.734970

Figure Lengend Snippet: Sub-cellular location of NDUFAF5. Human 143B cells were transfected with a plasmid encoding tagged NDUFAF5, and immunocytometry was performed 24 h later. Part A , cell nucleus stained with DAPI ( blue ); part B , recombinant NDUFAF5 detected with an anti-FLAG antibody, plus goat anti-mouse Alexa Fluor 488 ( green ); part C , mitochondria stained with MitoTracker ( red ); part D , merged areas of parts A–C .

Article Snippet: Human 143B osteosarcoma cells (ATCC number CRL8303) were grown at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM) containing 25 m m glucose and supplemented with fetal calf serum (10% v / v ), penicillin (100 units/ml), and streptomycin (0.1 mg/ml) under an atmosphere of 5% carbon dioxide.

Techniques: Transfection, Plasmid Preparation, Staining, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I *

doi: 10.1074/jbc.M116.734970

Figure Lengend Snippet: Suppression of expression of NDUFAF5 and assembly of complex I. Human 143B cells were transfected three times with either negative control siRNA or siRNA specific for NDUFAF5, denoted by a and b , respectively, and samples of mitoplasts and inner membranes were made 48 h after each transfection. Parts A and B , mitoplasts and inner mitochondrial membrane proteins, respectively, fractionated, in A by SDS-PAGE and, in B , by BN-PAGE. In part A , samples taken from three duplicate gels were Western-blotted with antibodies against subunits NDUFS2, NDUFS7, and ND1 of complex I, and one of them, employed as a loading control, was stained with Coomassie Blue dye, after Western blot analysis; in part B , membranes were probed with antibodies against the peripheral arm subunit NDUFS2, and membrane arm subunit NDUFB8, and probed a second time with an antibody against complex II subunit SDHB as a loading control, shown for the 192 h sample. CI-980 kDa , mature complex I; CI-200 kDa , −370 kDa, −550 kDa, sub-complexes of complex I; CII-130 kDa , complex II. The control cell results (denoted by the letter a in A and B ) have been presented previously in another context .

Article Snippet: Human 143B osteosarcoma cells (ATCC number CRL8303) were grown at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM) containing 25 m m glucose and supplemented with fetal calf serum (10% v / v ), penicillin (100 units/ml), and streptomycin (0.1 mg/ml) under an atmosphere of 5% carbon dioxide.

Techniques: Expressing, Transfection, Negative Control, Membrane, SDS Page, Western Blot, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I *

doi: 10.1074/jbc.M116.734970

Figure Lengend Snippet: Effect of transient suppression of expression of NDUFB3 and NDUFAF5 on the assembly of complex I. Samples were prepared at 192 h from human 143B cells that had been transfected three times at 72-h intervals with ( a ), negative control siRNA or ( b and c ), siRNA specific for NDUFB3 and NDUFAF5, respectively. Inner mitochondrial membrane proteins were fractionated by BN-PAGE, Western-blotted and probed with antibodies against the peripheral arm subunit NDUFS2 and assembly factor NDUFAF3. Antibodies against complex III (UQCRC1) and complex IV (COX5B) were used as loading controls on NDUFS2 and NDUFAF3 blots, respectively. CI , complex I; CIII 2 , complex III dimer; CIV , complex IV; CIII 2 +CIV , assembly of complex III dimer and complex IV; CI-200 kDa , complex I sub-complex.

Article Snippet: Human 143B osteosarcoma cells (ATCC number CRL8303) were grown at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM) containing 25 m m glucose and supplemented with fetal calf serum (10% v / v ), penicillin (100 units/ml), and streptomycin (0.1 mg/ml) under an atmosphere of 5% carbon dioxide.

Techniques: Expressing, Transfection, Negative Control, Membrane, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I *

doi: 10.1074/jbc.M116.734970

Figure Lengend Snippet: Suppression of expression of NDUFAF5 and oxygen consumption. Expression of NDUFAF5 in 143B cells was suppressed three times with siRNA at 72 h intervals. Part A , effect on levels of transcripts for control and NDUFAF5-depleted cells (white and hatched histograms, respectively) 48 h after each suppression. The values are normalized to endogenous β-actin, and error bars show the standard deviation. Parts B and C , oxygen consumption rates ( OCR ) of negative control and NDUFAF5 siRNA-treated cells ( white and shaded histograms , respectively) 120 h ( B ) and 192 h ( C ) after the first transfection, respectively. OCR was normalized to cell number. Rates were measured after successive additions of 2-deoxyglucose, rotenone, duroquinol, and antimycin, or 2-deoxyglucose, oligomycin, carbonylcyanide p -(trifluoromethoxy)-phenylhydrazone (FCCP), and a combination of rotenone and antimycin A. The OCR for complex I represents the rate for 2-deoxyglucose-treated cells minus the rate for rotenone-treated cells, and those for complex III are the OCR values for duroquinol minus antimycin A. The E/L ratio (FCCP/oligomycin) is an index of the maximum oxygen consumption capacity of the electron transport system ( E ) relative to the magnitude of uncoupled respiration ( L ).

Article Snippet: Human 143B osteosarcoma cells (ATCC number CRL8303) were grown at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM) containing 25 m m glucose and supplemented with fetal calf serum (10% v / v ), penicillin (100 units/ml), and streptomycin (0.1 mg/ml) under an atmosphere of 5% carbon dioxide.

Techniques: Expressing, Control, Standard Deviation, Negative Control, Transfection

Journal: Oncology Research

Article Title: Doxorubicin Inhibits Proliferation of Osteosarcoma Cells Through Upregulation of the Notch Signaling Pathway

doi: 10.3727/096504015X14343704124340

Figure Lengend Snippet: Effect of various concentrations and treatment time of doxorubicin on the proliferation of osteosarcoma cells. 143B cells were treated with 0.1, 0.5, 1, or 10 µM doxorubicin for 24, 48, and 72 h, and the proliferation of osteosarcoma cells was measured by MTT method. Data are expressed as mean ± SD of three independent experiments in triplicate. * p < 0.05, ** p < 0.01, or p > 0.05 versus control.

Article Snippet: The human osteosarcoma cell lines 143B obtained from the China Center for Type Culture Collection (CCTCC; Wuhan, China) were cultured in RPMI-1640 containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA).

Techniques: Control

Journal: Oncology Research

Article Title: Doxorubicin Inhibits Proliferation of Osteosarcoma Cells Through Upregulation of the Notch Signaling Pathway

doi: 10.3727/096504015X14343704124340

Figure Lengend Snippet: Effect of various concentrations and treatment time of doxorubicin on the apoptosis of osteosarcoma cells. 143B cells were treated with 0.1, 0.5, 1, or 10 µM doxorubicin for 24, 48, and 72 h, and mRNA expression (A) and activity (B) of caspase 3 in osteosarcoma cells were determined by RT-PCR and caspase 3 kit. Data are expressed as mean ± SD of three independent experiments in triplicate. * p < 0.05, ** p < 0.01, or p > 0.05 versus control.

Article Snippet: The human osteosarcoma cell lines 143B obtained from the China Center for Type Culture Collection (CCTCC; Wuhan, China) were cultured in RPMI-1640 containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA).

Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Oncology Research

Article Title: Doxorubicin Inhibits Proliferation of Osteosarcoma Cells Through Upregulation of the Notch Signaling Pathway

doi: 10.3727/096504015X14343704124340

Figure Lengend Snippet: Effect of various concentrations of doxorubicin on the activation of Notch signaling pathway in osteosarcoma cells. 143B cells were treated with 0.1, 0.5, 1, or 10 µM doxorubicin for 24, 48, and 72 h, and mRNA expression level of NOTCH1 (A), HEY1 (B), HES1 (C), and HES5 (D) was detected by real-time PCR. Data are expressed as mean ± SD of three independent experiments in triplicate. * p < 0.05, ** p < 0.01, or p > 0.05 versus control.

Article Snippet: The human osteosarcoma cell lines 143B obtained from the China Center for Type Culture Collection (CCTCC; Wuhan, China) were cultured in RPMI-1640 containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Control

Journal:

Article Title: Modulation of Gamma Interferon-Induced Major Histocompatibility Complex Class II Gene Expression by Porphyromonas gingivalis Membrane Vesicles

doi: 10.1128/IAI.70.3.1185-1192.2002

Figure Lengend Snippet: Inhibition of class II HLA-DRα surface expression in IFN-γ-inducible cells by P. gingivalis membrane vesicles. HUVECs, 143B osteosarcoma cells, MRC-5 fibroblasts, and THP-1 monocytic cells were treated with vesicles (OMVs, 30 μg of protein/ml) alone or IFN-γ (250 U/ml) in the absence or presence of vesicles at 37°C for 3 days. Control cells received no treatment. Cells were stained with either phycoerythrin-conjugated mouse anti-HLA-DR MAb or irrelevant isotype-matched MAb. Stained cells were analyzed by flow cytometry. Averages and standard deviations were calculated from three separate experiments.

Article Snippet: Other cell lines used, obtained from ATCC, included human embryonic lung fibroblasts (MRC-5; ATCC CCL 171), 143B human osteosarcoma cells (ATCC CRL 8304), and THP-1 monocytic cells (ATCC TIB 202).

Techniques: Inhibition, Expressing, Membrane, Control, Staining, Flow Cytometry

Journal: Cells

Article Title: Ischemic Stroke Risk Associated with Mitochondrial Haplogroup F in the Asian Population

doi: 10.3390/cells9081885

Figure Lengend Snippet: HIF-1α expressions in different cybrids under hypoxia-ischemia. Comparison of HIF-1α expression under hypoxic-ischemic conditions in cybrids harboring common mitochondrial haplogroups found in the ethnic Chinese population (B4, B5, D4, D5, F1, F2, N9). The 143B cybrid was used to represent the Caucasian population. Actin was used as a loading control. Data represent the mean ± SD of at least three independent experiments ( # p < 0.005). N was used to represent normoxic and H to represent the hypoxic condition for 24 h.

Article Snippet: MtDNA-depleted ρ0 cells were established by the treatment of human osteosarcoma 143B cells (BCRC 60439) (Bioresource Collection and Research Center, Taipei, Taiwan) with ethidium bromide for three months, and illustration of their mtDNA-less cellular status by RT-PCR has been previously described [ , ].

Techniques: Comparison, Expressing, Control