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

Journal: Cell Reports Medicine

Article Title: GIPR antagonist antibodies conjugated to GLP-1 peptide are bispecific molecules that decrease weight in obese mice and monkeys

doi: 10.1016/j.xcrm.2021.100263

Figure Lengend Snippet: Co-localization of hGIPR-Ab/P1 with hGLP-1R and hGIPR receptors and with early and recycling endosome markers U2OS cells stably expressing SNAP-tagged hGLP-1R and hGIPR were fixed and permeabilized at indicated time points after hGIPR-Ab/P1 stimulation. (A) hGIPR-Ab/P1 (green) co-localized with hGLP-1R (orange) and hGIPR (red) in dual receptor-expressing cells (30 min after stimulation). (B) hGIPR-Ab/P1 (green) co-localized with hGLP-1R (orange) and EEA1 (red), an early endosome marker. (C) hGIPR-Ab/P1 (green) co-localized with hGLP-1R (orange) and Rab11 (red), a perinuclear recycling endosome marker. Data shown are representative of n ≥ 3 experiments.

Article Snippet: U2OS SNAP-tagged hGLP-1R + hGIPR, clone 16-1 , Amgen , N/A.

Techniques: Stable Transfection, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: GIPR antagonist antibodies conjugated to GLP-1 peptide are bispecific molecules that decrease weight in obese mice and monkeys

doi: 10.1016/j.xcrm.2021.100263

Figure Lengend Snippet:

Article Snippet: U2OS SNAP-tagged hGLP-1R + hGIPR, clone 16-1 , Amgen , N/A.

Techniques: Clinical Proteomics, Recombinant, Control, Protease Inhibitor, DC Protein Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Gene Expression, Stable Transfection, Expressing, Software, Imaging, Real-time Polymerase Chain Reaction

Journal: Biomedicines

Article Title: Anti-Inflammatory and Cancer-Preventive Potential of Chamomile ( Matricaria chamomilla L.): A Comprehensive In Silico and In Vitro Study

doi: 10.3390/biomedicines12071484

Figure Lengend Snippet: Confocal immunofluorescence microscopy of the microtubule network in U2OS cells upon treatment with ( A ) quercetin and ( B ) lupeol at concentrations of 0.1 µM, 1 µM, and 10 µM for 24 h. Vincristine (1 µM) and paclitaxel (1 µM) served as positive controls and DMSO as the negative control. The cells were imaged using a Thunder Imager Live Cell microscope with a 63×/1.40 NA objective lens (HC PL APO CS2 63×/1.40 OIL UV). The microtubules were visualized using green fluorescence for GFP (green), and the images were merged with DAPI (blue) to highlight the nucleus.

Article Snippet: In this context, it was interesting that the cytotoxicity of both compounds in CCRF-CEM and U2OS cells was not caused by apoptosis as the most common mode of cell death but by necrosis.

Techniques: Immunofluorescence, Microscopy, Negative Control, Fluorescence

Journal: Biomedicines

Article Title: Anti-Inflammatory and Cancer-Preventive Potential of Chamomile ( Matricaria chamomilla L.): A Comprehensive In Silico and In Vitro Study

doi: 10.3390/biomedicines12071484

Figure Lengend Snippet: Cell cycle arrest of U2OS cells by quercetin and lupeol. ( A ) Debris was gated out (SSC-A vs. FSC-A) with the first gate. ( B ) With the second gate (FSC-H vs. FSC-A), only single cells of normal morphology were gated. Duplets were gated out. ( C , D ) Three-dimensional representation of DNA histograms of U2OS cells exposed to 1 × IC 50 and 4 × IC 50 quercetin and lupeol for 72 h. DMSO was used as the negative control, and 1 × IC 50 vincristine was used as the positive control. ( C ) Cells treated with lupeol and ( D ) cells treated with quercetin. The histograms were obtained through flow cytometry using an excitation of 488 nm and an emission wavelength of 530 nm. ( E , F ) Bar diagrams showing the distinct phases of cell cycle upon treatment with quercetin and lupeol for 72 h. ( E ) Cells treated with lupeol and ( F ) cells treated with quercetin. The bar diagrams were created through the calculation of the mean values ± SD of three independent experiments. *** p < 0.001, ** p < 0.01, and * p < 0.05 compared to the negative control using paired two-tailed t -test.

Article Snippet: In this context, it was interesting that the cytotoxicity of both compounds in CCRF-CEM and U2OS cells was not caused by apoptosis as the most common mode of cell death but by necrosis.

Techniques: Negative Control, Positive Control, Flow Cytometry, Two Tailed Test

Journal: Biomedicines

Article Title: Anti-Inflammatory and Cancer-Preventive Potential of Chamomile ( Matricaria chamomilla L.): A Comprehensive In Silico and In Vitro Study

doi: 10.3390/biomedicines12071484

Figure Lengend Snippet: Dose–response curves of quercetin and lupeol as determined through resazurin assay. The mean values and standard deviation values are from three independent experiments. The tumor cells were subjected to treatment with each compound at concentrations of 10 µM, 25 µM, 50 µM, and 100 µM for 72 h. ( A ) Sensitive CCRF-CEM and the drug-resistant P-glycoprotein overexpressing CEM/ADR5000 leukemia cells. ( B ) U87/ΔEGFR transfected with a deletion-activated cDNA of EGFR and its wild-type U87MG glioblastoma cells. ( C ) HCT116 p53 +/+ and knockout HCT116 p53 −/− colorectal cancer cells. ( D ) U2OS osteosarcoma cells.

Article Snippet: In this context, it was interesting that the cytotoxicity of both compounds in CCRF-CEM and U2OS cells was not caused by apoptosis as the most common mode of cell death but by necrosis.

Techniques: Resazurin Assay, Standard Deviation, Transfection, Knock-Out

Journal: Biomedicines

Article Title: Anti-Inflammatory and Cancer-Preventive Potential of Chamomile ( Matricaria chamomilla L.): A Comprehensive In Silico and In Vitro Study

doi: 10.3390/biomedicines12071484

Figure Lengend Snippet: Detection of cell death in U2OS cells using flow cytometry and annexin-V/PI staining using a flow cytometer. ( A , B ) Cells treated for 72 h with 0.5 × IC 50 , 1 × IC 50 , 2 × IC 50 , and 4 × IC 50 of lupeol and quercetin, respectively. ( C , D ) represent bar diagrams of the percentage of cells in quadrium treated with lupeol ( C ) and quercetin ( D ). For details, see . Treatment with both compounds at 4 × IC 50 showed a tendency for increased necrosis, which was, however, not statistically significant compared to the negative control.

Article Snippet: In this context, it was interesting that the cytotoxicity of both compounds in CCRF-CEM and U2OS cells was not caused by apoptosis as the most common mode of cell death but by necrosis.

Techniques: Flow Cytometry, Staining, Negative Control

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: HBB2 robustly induces the heat shock response. ( a ) Chemical structures of HBB2 and DBA. ( b ) Luciferase gene reporter assay was performed in HeLa-luc cells treated with up to 5 µM of either HBB2 or DBA for 24 h. PEITC was included as a positive control. Luminescence values were normalised to respective vehicle controls (0.1% DMSO for HBB2 and DBA; 0.1% ACN (not included in the graph) for PEITC). Data are means ± SD from two independent experiments (n = 6 for each treatment group). *** p < 0.001 (one-way ANOVA and Tukey’s post-test). ( c ) Western blot analysis demonstrating a concentration-dependent increase in total Hsp70 levels in U2OS whole-cell lysates caused by a 24-h treatment with HBB2 but not with DBA. 0.1% DMSO was used as a vehicle control and β-actin acted as a loading control.

Article Snippet: Human osteosarcoma U2OS cells, a kind gift from Sonia Rocha (School of Life Sciences, University of Dundee, UK), were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Life Technologies) that contains L-glutamine, sodium pyruvate, and high D-glucose content (4.5 g/L), supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS; Thermo Scientific).

Techniques: Luciferase, Reporter Assay, Positive Control, Western Blot, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: HBB2 causes lipidation of LC3B and accumulation of p62, and enhances autophagic flux. ( a ) Immunofluorescence analysis for p62 and LC3B in U2OS cells that had been exposed to HBB2 (3 µM) (bottom) or treated with vehicle (top) for 16 h. The images were obtained by confocal microscopy. Scale bar = 20 µm. ( b–d ) Western blot analysis of U2OS whole-cell lysates was conducted to detect the changes in the levels of LC3B-II ( b ), Hsp70 ( c ) and p62 (d) in response to a 4-, 8-, 16-, or 24 h treatments with HBB2 (3 µM) and/or bafilomycin A1 (Baf-A1; 10 nM), as indicated above the lanes. DMSO (0.1%, v/v) was used as a vehicle control (indicated as −/− above the blots), whereas β-actin levels served as a loading control.

Article Snippet: Human osteosarcoma U2OS cells, a kind gift from Sonia Rocha (School of Life Sciences, University of Dundee, UK), were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Life Technologies) that contains L-glutamine, sodium pyruvate, and high D-glucose content (4.5 g/L), supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS; Thermo Scientific).

Techniques: Immunofluorescence, Confocal Microscopy, Western Blot, Control

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: Knockdown of NRF2 inhibits autophagy. ( a ) Immunoblotting analysis of NRF2, p62 and LC3B in lysates from U2OS cells, which had been transfected with either control siRNA (siCTL) or NRF2 siRNA (siNRF2) for 48 h, and subsequently treated with vehicle (0.1% DMSO) or HBB2 (3 µM) for 18 h, and supplemented with 10 nM bafilomycin A1 (Baf-A1) or vehicle (0.1% DMSO) for the last 2 h. The levels of β-actin served as a loading control. For detection of LC3B, proteins from cell lysates were resolved using 13% SDS-PAGE and transferred onto 0.45 µm PVDF membranes, whereas for detection of NRF2 and actin, proteins were blotted onto 0.45 µm NC membranes. ( b–d ) Real-time PCR analysis of NRF2 ( b ), HSF1 ( c ), and p62 ( d ) from a parallel experiment described in ( a ) except without Baf-A1 treatment. The mRNA levels of 18 S were used for normalization. The data are represented by mean + SD from three independent transfections. Student’s t-test was used to test for statistical significance, where *represents p < 0.05 when comparing siCTL and siNRF2 for each treatment pair.

Article Snippet: Human osteosarcoma U2OS cells, a kind gift from Sonia Rocha (School of Life Sciences, University of Dundee, UK), were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Life Technologies) that contains L-glutamine, sodium pyruvate, and high D-glucose content (4.5 g/L), supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS; Thermo Scientific).

Techniques: Knockdown, Western Blot, Transfection, Control, SDS Page, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: NRF2 is required for basal and HBB2-induced autophagic flux. Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868). Wide-field microscope (Deltavision Elite) was used to collect the images where in each field 25 images were taken with an optical section of 0.2 μm. The deconvolved images represent the summed intensity projection using SoftWorX (Version 5.5). Scale bar = 20 µm.

Article Snippet: Human osteosarcoma U2OS cells, a kind gift from Sonia Rocha (School of Life Sciences, University of Dundee, UK), were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Life Technologies) that contains L-glutamine, sodium pyruvate, and high D-glucose content (4.5 g/L), supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS; Thermo Scientific).

Techniques: Immunofluorescence, Transfection, Control, Microscopy

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: HSF1 inhibits autophagic flux. ( a ) Immunoblotting analysis of HSF1, Hsp70, p62, mTOR, phosphorylated mTOR (pS2448), p70 S6K, phosphorylated p70 S6K (pT389) in lysates from U2OS cells, which had been transfected with either control siRNA (siCTL) or HSF1 siRNA (siHSF1s or siHSF1sp) for 48 h, and subsequently treated with vehicle (0.1% DMSO) or HBB2 (3 µM) for 18 h, and supplemented with bafilomycin A1 (Baf-A1; 10 nM) or vehicle (0.1% DMSO) for a the last two hours of HBB2 treatment. The levels of β -actin served as a loading control. ( b–d ) Real-time PCR analysis of HSF1 ( b ), NRF2 ( c ), and p62 ( d ) from a parallel experiment described in ( a ) except without Baf-A1 treatment, in U2OS cells transfected with siCTL (10 nM) or siHSF1sp (10 nM). The mRNA levels of 18 S were used for normalization. The data are represented by means + SD from three independent transfections. Student’s t-test was used to test for statistical significance, where *represents p < 0.05 when comparing siCTL and siHSF1sp for each treatment pair. ( e ) Immunoblotting analysis of LC3B in lysates from U2OS cells, which had been transfected with either siCTL (10 nM) or siHSF1sp siRNA (10 nM) for 48 h, and subsequently treated with vehicle (0.1% DMSO) or HBB2 (3 µM) for 18 h, supplemented with bafilomycin A1 (Baf-A1; 10 nM) for the last 2 h. The levels of β-actin served as a loading control.

Article Snippet: Human osteosarcoma U2OS cells, a kind gift from Sonia Rocha (School of Life Sciences, University of Dundee, UK), were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Life Technologies) that contains L-glutamine, sodium pyruvate, and high D-glucose content (4.5 g/L), supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS; Thermo Scientific).

Techniques: Western Blot, Transfection, Control, Real-time Polymerase Chain Reaction