Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Cell morphology was observed with a light microscope (A) . Cell proliferation was analyzed using a trypan blue exclusion assay (B) . After drug administration, cellular proteins were prepared for immunoblot analyses. Levels of ERα and ERβ were immunodetected (C and E, top panels) . Amounts of β-actin were analyzed as the internal standard (C and E, bottom panels) . These protein bands were quantified and statistically analyzed (D and F) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the values significantly ( p < 0.05) differed from the respective control group, p < 0.05.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Light Microscopy, Trypan Blue Exclusion Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Cellular nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (middle panel). The merged signals indicated that the ERα protein had been translocated into nuclei (bottom panel). These merged fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p < 0.05.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Staining, Control

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were treated with 10 nM of estradiol for 48 h. Total RNA were isolated for analysis of mitochondrial energy metabolism genes using a PCR array, containing 84 genomic genes encoding certain mitochondrial enzymes for ATP synthesis and 12 loading controls (A) . Differential expressions of these genes were measured and shown as a hot map in the order of genes indicated in panel A (B) . Percentages of upregulated, downregulated, and unchanged expressions of these genes were further statistically analyzed (C) . Also, the major genomic complex genes upregulated by estradiol in human osteoblasts were summarized (D) .

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Isolation

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Mitochondria of human osteoblasts were stained with 3,3′-dihexyloxacarbocyanine (DiOC6), a positively charged dye (middle panel). Merged signals indicated that the ERα protein had been translocated into mitochondria (bottom panels). These fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p < 0.05.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Staining, Control

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 3, 6, 12, 18, and 24 h. Levels of COX I and II mRNA were analyzed using an RT-PCR ( A and C , top panels). Amounts of β-actin mRNA were assayed as the internal standard (bottom panel). These bands were quantified and statistically analyzed (B and D) . A quantitative real-time PCR analysis was carried out to confirm expression of COX I mRNA in U2OS cells and rat calvarial osteoblasts (E) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p < 0.05.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Levels of COX I and II proteins were immunodetected (A and C, top panels) . Amounts of β-actin were analyzed as the internal standard (A and C, bottom panels) . These protein bands were quantified and statistically analyzed ( B and D ). Mitochondria of human osteoblasts were stained with 3,3′-dihexyloxacarbocyanine (DiOC6), a positively charged dye. The mitochondrial membrane potential (MMP) of human osteoblasts was determined by quantifying DiOC6-positive signals (E) . The mitochondrial enzyme activity was assayed using a colorimetric method (F) . Cellular ATP levels were quantified using a bioluminescence assay (G) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p < 0.05. FI, fluorescent intensity.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Staining, Membrane, Activity Assay, ATP Bioluminescent Assay, Control

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were treated with ERα siRNA for 24 and 48 h. Scrambled siRNA was administered to control cells as the negative standard. Levels of ERα were immunodetected ( A , top panel). Amounts of β-actin were analyzed as the internal standard (bottom panel). These protein bands were quantified and statistically analyzed (B) . After knocking-down ERα translation for 24 h, human osteoblasts were treated with estradiol for another 6 h. A quantitative PCR analysis was conducted to determine COX I mRNA expression (C) . Each value represents the mean ± SEM, n = 3. The symbols * and # indicate that a value significantly ( p < 0.05) differed from the control and estradiol-treated groups, respectively.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

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

Journal: Materials

Article Title: The Biocompatibility and Self-Healing Effect of a Biopolymer’s Coating on Zn Alloy for Biomedical Applications

doi: 10.3390/ma16237486

Figure Lengend Snippet: Confocal images of U2OS cells with DAPI-stained nucleus after 7 days of incubation on casein-based layer (2cas1h) on tested materials: ZnMg3.2 alloy ( a ); casein coating (2cas1h) ( b ). Additional 3D visualization from Immaris software (Bitplane Scientific Software; version 10.0.0; Olympus, Zurich, Switzerland) for ZnMg3.2 alloy ( c ), casein coating (2cas1h) ( d ).

Article Snippet: Biological characterization was based on a human osteosarcoma cell line U2OS (cat. No. 92022711; Merck, Rahway, NJ, USA).

Techniques: Staining, Incubation, Software

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