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:

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: Cancer Management and Research

Article Title:

Circular RNA hsa_circ_0003496 Contributes to Tumorigenesis and Chemoresistance in Osteosarcoma Through Targeting (microRNA) miR-370/Krüppel-Like Factor 12 Axis

doi: 10.2147/cmar.s253969

Figure Lengend Snippet: Figure 2 Circ_0003496 depletion repressed OS/DXR cell proliferation, migration and invasion and enhanced apoptosis and DXR sensitivity. KHOS/DXR and MG63/DXR cells were transfected with si-NC or si-circ_0003496, followed by the measurement of circ_0003496 expression by qRT-PCR (A), the IC50 value for DXR (B) and cell proliferation (C and D) by CCK-8 assay, cell migration (E) and invasion (F) by transwell assay, cell apoptosis by flow cytometry (G), the expression levels of MRP1, P-gp and LRP by Western blot (H). The scale represented 100 µm. *P < 0.05.

Article Snippet: Human KHOS (ATCC®CRL-1544, American Type Culture Collection, ATCC, Manassas, VA, USA) and MG63 (ATCC®CRL-1427, ATCC) OS cell lines were maintained in a humidified atmosphere containing 5% CO2 in Eagle’s Minimum Essential Medium (ATCC) plus 10% fetal bovine serum (Lonza, Basel, Switzerland) at 37°C.

Techniques: Migration, Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Transwell Assay, Flow Cytometry, Western Blot

Journal: Cancer Management and Research

Article Title:

Circular RNA hsa_circ_0003496 Contributes to Tumorigenesis and Chemoresistance in Osteosarcoma Through Targeting (microRNA) miR-370/Krüppel-Like Factor 12 Axis

doi: 10.2147/cmar.s253969

Figure Lengend Snippet: Figure 1 Circ_0003496 overexpression was associated with DXR resistance of OS. (A) Circ_0003496 expression was assessed by qRT-PCR in OS tissues from 35 primary patients (Chemosensitive tissues) and 35 recurrent patients (Chemoresistant tissues). (B–D) KHOS/DXR and MG63/DXR cells were exposed to various concentrations (0, 1, 2, 4, 8, 16, 32, 64 and 128 µg/mL) of DXR for 48 h, followed by the detection of cell viability by CCK-8 assay. (E) Circ_0003496 expression was evaluated by qRT-PCR in KHOS, MG63, KHOS/DXR and MG63/DXR cells. *P < 0.05.

Article Snippet: Human KHOS (ATCC®CRL-1544, American Type Culture Collection, ATCC, Manassas, VA, USA) and MG63 (ATCC®CRL-1427, ATCC) OS cell lines were maintained in a humidified atmosphere containing 5% CO2 in Eagle’s Minimum Essential Medium (ATCC) plus 10% fetal bovine serum (Lonza, Basel, Switzerland) at 37°C.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, CCK-8 Assay

Journal: Cancer Management and Research

Article Title:

Circular RNA hsa_circ_0003496 Contributes to Tumorigenesis and Chemoresistance in Osteosarcoma Through Targeting (microRNA) miR-370/Krüppel-Like Factor 12 Axis

doi: 10.2147/cmar.s253969

Figure Lengend Snippet: Figure 3 Circ_0003496 acted as a molecular sponge of miR-370. (A) Schematic model of the miR-370-binding sequence in circ_0003496 and mutated miR-370-binding sites. (B and C) Relative luciferase activity was detected in KHOS/DXR and MG63/DXR cells cotransfected with circ_0003496 wild-type reporter construct (circ_0003496 WT) or circ_0003496 mutant-type reporter construct (circ_0003496 MUT) and miR-370 mimic or miR-NC mimic. (D) KHOS/DXR and MG63/DXR cells transfected with miR-370 mimic or miR-NC mimic were lysed and incubated with beads-conjugated anti-Ago2 or anti-IgG antibody, followed by the determination of circ_0003496 enrichment by qRT-PCR. (E and F) Cell lysates of KHOS/DXR and MG63/DXR cells were incubated with Bio-NC, wild-type Bio-circ_0003496 (Bio-circ_0003496 WT) or mutant-type Bio-circ_0003496 (Bio-circ_0003496 MUT), and then miR-370 enrichment was assessed by qRT-PCR. MiR-370 expression was detected by qRT-PCR in 35 primary patients (Chemosensitive tissues) and 35 recurrent patients (Chemoresistant tissues) (G), KHOS, MG63, KHOS/DXR and MG63/DXR cells (H), KHOS/DXR and MG63/DXR cells transfected with pcDNA or circ_0003496 overexpression plasmid (I). *P < 0.05.

Article Snippet: Human KHOS (ATCC®CRL-1544, American Type Culture Collection, ATCC, Manassas, VA, USA) and MG63 (ATCC®CRL-1427, ATCC) OS cell lines were maintained in a humidified atmosphere containing 5% CO2 in Eagle’s Minimum Essential Medium (ATCC) plus 10% fetal bovine serum (Lonza, Basel, Switzerland) at 37°C.

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Construct, Mutagenesis, Transfection, Incubation, Quantitative RT-PCR, Expressing, Over Expression, Plasmid Preparation

Journal: Cancer Management and Research

Article Title:

Circular RNA hsa_circ_0003496 Contributes to Tumorigenesis and Chemoresistance in Osteosarcoma Through Targeting (microRNA) miR-370/Krüppel-Like Factor 12 Axis

doi: 10.2147/cmar.s253969

Figure Lengend Snippet: Figure 4 MiR-370 mediated the regulatory effect of circ_0003496 knockdown on OS/DXR cell proliferation, migration, invasion and DXR sensitivity. KHOS/DXR and MG63/DXR cells were transfected with si-NC, si-circ_0003496, si-circ_0003496+in-miR-NC or si-circ_0003496+in-miR-370, followed by the assessment of miR-370 expression by qRT-PCR (A), the IC50 value for DXR (B) and cell proliferation (C and D) by CCK-8 assay, cell migration (E) and invasion (F) by transwell assay, cell apoptosis by flow cytometry (G), the expression levels of MRP1, P-gp and LRP by Western blot (H). *P < 0.05.

Article Snippet: Human KHOS (ATCC®CRL-1544, American Type Culture Collection, ATCC, Manassas, VA, USA) and MG63 (ATCC®CRL-1427, ATCC) OS cell lines were maintained in a humidified atmosphere containing 5% CO2 in Eagle’s Minimum Essential Medium (ATCC) plus 10% fetal bovine serum (Lonza, Basel, Switzerland) at 37°C.

Techniques: Knockdown, Migration, Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Transwell Assay, Flow Cytometry, Western Blot

Journal: Cancer Management and Research

Article Title:

Circular RNA hsa_circ_0003496 Contributes to Tumorigenesis and Chemoresistance in Osteosarcoma Through Targeting (microRNA) miR-370/Krüppel-Like Factor 12 Axis

doi: 10.2147/cmar.s253969

Figure Lengend Snippet: Figure 5 Circ_0003496 protected against KLF12 repression through acting as a miR-370 sponge. (A) Schematic of the target sequence for miR-370 within KLF12 3ʹ-UTR and the mutant in the target region. (B and C) Relative luciferase activity was detected in KHOS/DXR and MG63/DXR cells cotransfected with KLF12 3ʹ-UTR wild-type reporter construct (KLF12 3ʹ-UTR WT) or KLF12 3ʹ-UTR mutant-type reporter construct (KLF12 3ʹ-UTR MUT) and miR-370 mimic or miR-NC mimic. (D and E) KLF12 expression was tested by qRT-PCR and Western blot in 35 primary patients (Chemosensitive tissues) and 35 recurrent patients (Chemoresistant tissues). KLF12 mRNA and protein levels were determined by qRT-PCR and Western blot in KHOS, MG63, KHOS/DXR and MG63/DXR cells (F and G), KHOS/DXR and MG63/DXR cells transfected with miR-370 mimic or miR-NC mimic (H and I). (J and K) KHOS/DXR and MG63/DXR cells were transfected with pcDNA, circ_0003496, circ_0003496+miR-NC mimic or circ_0003496+miR-370 mimic, followed by the measurement of KLF12 by qRT-PCR and Western blot. circ_0003496: circ_0003496 overexpression plasmid. *P < 0.05.

Article Snippet: Human KHOS (ATCC®CRL-1544, American Type Culture Collection, ATCC, Manassas, VA, USA) and MG63 (ATCC®CRL-1427, ATCC) OS cell lines were maintained in a humidified atmosphere containing 5% CO2 in Eagle’s Minimum Essential Medium (ATCC) plus 10% fetal bovine serum (Lonza, Basel, Switzerland) at 37°C.

Techniques: Sequencing, Mutagenesis, Luciferase, Activity Assay, Construct, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Over Expression, Plasmid Preparation

Journal: Cancer Management and Research

Article Title:

Circular RNA hsa_circ_0003496 Contributes to Tumorigenesis and Chemoresistance in Osteosarcoma Through Targeting (microRNA) miR-370/Krüppel-Like Factor 12 Axis

doi: 10.2147/cmar.s253969

Figure Lengend Snippet: Figure 6 MiR-370 up-regulation exerted its regulatory effects on OS/DXR cell proliferation, migration, invasion and DXR sensitivity by KLF12. KHOS/DXR and MG63/DXR cells were transfected with miR-370 mimic, miR-NC mimic, miR-370 mimic+pcDNA or miR-370 mimic+KLF12, followed by the detection of KLF12 expression by qRT-PCR and Western blot (A and B), the IC50 value for DXR (C) and cell proliferation (D and E) by CCK-8 assay, cell migration (F) and invasion (G) by transwell assay, cell apoptosis by flow cytometry (H), the expression levels of MRP1, P-gp and LRP by Western blot (I). KLF12: KLF12 overexpression plasmid. *P < 0.05.

Article Snippet: Human KHOS (ATCC®CRL-1544, American Type Culture Collection, ATCC, Manassas, VA, USA) and MG63 (ATCC®CRL-1427, ATCC) OS cell lines were maintained in a humidified atmosphere containing 5% CO2 in Eagle’s Minimum Essential Medium (ATCC) plus 10% fetal bovine serum (Lonza, Basel, Switzerland) at 37°C.

Techniques: Migration, Transfection, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Transwell Assay, Flow Cytometry, Over Expression, Plasmid Preparation