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: Cancer Immunology, Immunotherapy : CII

Article Title: Osteosarcoma cell–derived CCL2 facilitates lung metastasis via accumulation of tumor-associated macrophages

doi: 10.1007/s00262-025-04051-x

Figure Lengend Snippet: CCL2-neutralizing antibody prevents lung metastasis via suppression of macrophage polarization in the lung. A , B Mouse LM8 cells ( A ) and human 143B cells ( B ) with metastatic potential (2 × 10 6 cells) were inoculated into the tibia of C3H/Hej mice and athymic nude mice, respectively. CCL2-neutralizing antibody (CCL2 MAB) and control isotype IgG (Iso MAB) (200 μg/mouse) were administered intraperitoneally twice per week, starting 3 days before inoculation. Lung metastases and the immune microenvironment in mice that received orthotopic inoculation with LM8 and 143B cells were assessed on days 21 and 28 after tumor inoculation, respectively. C , D Left panel: representative photographs of lung tissues stained with hematoxylin/eosin at low magnification. Right panel: number of metastatic nodules in the lung tissues is shown as mean ± SD (n = 5 in each group; *, P < 0.05; **, P < 0.01; ns, not significant). E , F Comparison of the polarization of macrophages in lung tissues. The percentages of M1-like and M2-like macrophages are shown as mean ± SD (n = 5 in each group; *, P < 0.05; ns, not significant). Statistical significance was determined using one-way ANOVA with Tukey’s post-test. Figures were generated using BioRender

Article Snippet: HOS and 143B cells (2 × 10 6 cells) were orthotopically inoculated into the proximal tibia of 6-week-old female athymic nude mice (CLEA Japan) (n = 4 in each group).

Techniques: Control, Staining, Comparison, Generated

Journal: Molecular Biomedicine

Article Title: Detection of alternative lengthening of telomeres mechanism on tumor sections

doi: 10.1186/s43556-021-00055-y

Figure Lengend Snippet: Establishment of an ALT + tumor xenograft model in NSG mice. Tumor volume was monitored over time with a digital caliper after injection of 10 6 cells in the flank of NSG mice. 143B//TEL + is an osteosarcoma cell line ( n = 3); HT1080/TEL + comes from a fibrosarcoma ( n = 5); LB857/ALT + is a myxoid sarcoma cell line ( n = 4); MG63/TEL + and LB188/ALT + derive from, respectively, an osteosarcoma and a rhabdomyosarcoma ( n = 2) tumor and SaOS/ALT + is an osteosarcoma cell line ( n = 1). Mean + SEM. Representative pictures of the xenografts are shown

Article Snippet: The osteosarcoma cell lines, SaOS-2/ALT + (ATCC, HTB-85), U2OS/ALT + (ATCC, HTB-96), MG63/TEL + (ATCC, CRL-1427) and 143B/TEL + (Coriell Institute for medical Research, New Jersey, USA) were grown in DMEM (Gibco) supplemented with 10% FBS (Gibco), 1% glutamine (Gibco) and 1% penicillin/streptomycin (PS) (Gibco).

Techniques: Injection