Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 1. TGF-β expression in osteosarcoma specimens and cell lines. (a) Immunohistochemical staining was used to analyze TGF-β expression in both osteosarcoma specimens and normal adjacent tissues. (b) Survival analysis was performed using the Kaplan-Meier method. Data are presented as mean ± SEM of three independent experiments. (c) TGF-β protein levels in osteosarcoma cell lines (*P < 0.05). (d) TGF-β mRNA expression in osteosarcoma cell lines (*P < 0.05).

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 2. Small interfering (si) RNA targeting of TGF-β (si-TGF-β) suppresses proliferation and promotes apoptosis in osteosarcoma. (a) RT-qPCR was employed to assess the knockdown efficiency of si-TGF-β, *P < 0.05 vs. the si-control group. (b) Western blotting was used to measure the knockdown efficiency of si-TGF-β, *P < 0.05 vs. the si-control group. (c)The MTT assay was used to assess the viability of U2OS and MG-63 cells transfected with si-control or si-TGF-β, *P < 0.05 vs. the si-control group. (d and e) The colony- forming ability of U2OS and MG-63 cells transfected with si-control or si-TGF -β, *P < 0.05 vs. the si-control group. (f and g) Flow cytometry analysis of the cell cycle phase distribution of U2OS and MG-63 cells transfected with si-control or si-TGF – β, *P < 0.05 vs. the si-control group. (h and i) The rate of apoptosis in U2OS and MG-63 cells transfected with si-TGF-β, *P < 0.05 vs. the si-control group. (j and k) TGF-β knockdown increases the expression of cleaved PARP, caspase-3, and BAX, *P < 0.05 vs. the si-control group.

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Quantitative RT-PCR, Knockdown, Control, Western Blot, MTT Assay, Transfection, Flow Cytometry, Expressing

Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 4. TGF-β enhances osteosarcoma cell stemness and increases the proportion of CD133+ cells. (a and b) Spheroid formation assay for U2OS and MG-63 cells transfected with si-TGF-β. Representative images (left panel) and statistical measurements (right panel). Representative micrographs of formed spheres were analyzed in cells treated with si-TGF-β or si-control. Scale bar, 100 μm. *P < 0.05 vs. the si-control group. (c and d) The percentage of CD133+ cells in U2OS- and MG-63-derived spheroids was analyzed by flow cytometry.*P < 0.05 vs. the si-control group. (e and f) Stem cell-associated protein expression was measured by western blot. *P < 0.05 vs. the si-control group.

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Tube Formation Assay, Transfection, Control, Derivative Assay, Flow Cytometry, Expressing, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 5. TGF-β regulates the PI3K/mTOR signaling pathway in osteosarcoma cells. (a) p-PI3K and p-Akt expression was measured by western blot, P < 0.05 vs. the si-control group. (b and c) The colony-forming ability of si-TGF-β-transfected U2OS and MG-63 cells with or without LY294002 treatment. P < 0.05 vs. the si-control group. (d and e) Transwell assays were used to assess the invasive ability of U2OS and MG-63 cells, P < 0.05 vs. the si-control group. (f and g) A wound-healing assay was performed to measure the migratory ability of U2OS and MG-63 cells, P < 0.05 vs. the si-control group. (h and i) Spheroid formation assay for U2OS/MG-63 cells transfected with si-TGF-β. Representative images (left panel) and statistical measurement (right panel). Representative micrographs of formed spheres were analyzed in cells treated with si-TGF-β or si-control with or without LY294002 treatment. Scale bar, 100 μm. *P < 0.05 vs. the si-control group. (j and k) The percentage of CD133+ cells in U2OS cell- and MG-63 cell-derived spheroids was analyzed by flow cytometry. *P < 0.05 vs. the si-control group.

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Expressing, Western Blot, Control, Transfection, Wound Healing Assay, Tube Formation Assay, Derivative Assay, 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

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