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

Journal: Nature Communications

Article Title: Direct single-molecule detection and super-resolution imaging with a low-cost portable smartphone-based microscope

doi: 10.1038/s41467-025-63993-z

Figure Lengend Snippet: a Sketch of an immunolabeled microtubule with primary and secondary antibodies. The secondary antibody is conjugated with a docking DNA strand complementary to the Cy3B-labeled imager strand. b Super-resolution and diffraction-limited images of the microtubule network of fixed U2OS cells obtained with the smartphone-based microscope. The super-resolved image represents the localization density, while the diffraction-limited image is the actual fluorescence intensity. Scale bar: 2 µm. c , d Comparison of super-resolved images of the microtubule network obtained with the smartphone-based setup ( c ) – a magnified image of the region marked in ( b ) - and with the high-end microscope ( d ). Scale bar: 2 µm. e Line profiles across super-resolved microtubules as indicated in ( c ) and ( d ). Black solid lines: smartphone-based microscope. Pink dashed lines: high-end microscope. In total, four different videos were obtained in which the immunolabeled microtubules could be resolved with the smartphone-based microscope. Source data are provided as a Source Data file.

Article Snippet: A human bone osteosarcoma epithelial (U2OS) cell culture with immunolabeled microtubules was purchased from Massive Photonics.

Techniques: Immunolabeling, Labeling, Microscopy, Fluorescence, Comparison