u2os cells Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    ATCC u2os cells
    RT53 induces unregulated necrotic cell death. (A) Ultrastructural analysis of RT53-mediated cell death. <t>U2OS</t> cells were left untreated (Control) or exposed to 15 μM of RT53 for 30 min. Cells were then analyzed by transmission electron microscopy following osmium tetroxide staining. (B) U2OS cells were exposed to 20 μM of RT53 in the presence or absence of 50 μM zVAD-fmk, 50 μM Necrostatin-1 (Nec-1) or 100 mM cyclosporin A (CsA) for 1 h. Necrotic cell death was monitored by lactate dehydrogenase (LDH) release from cells into the culture medium. The obtained values were normalized to those of the maximum LDH released (completely lysed) control. Data are means±s.e.m. ( n = 3).
    U2os Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2589 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u2os cells/product/ATCC
    Average 99 stars, based on 2589 article reviews
    Price from $9.99 to $1999.99
    u2os cells - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore u2os cells
    Two direct circadian modulators, KL001 (A) and PF-670462 (B), and three indirect circadian modulators, Chir99021 (C), etoposide (D), and SP600125 (E), influence cell motility to various extents. KL001 at concentrations 5, 7, and 12 µM promotes cell motility at T = 6, 12, and 18 hours (A). PF-670462 at 1.5 µM concentration reduces cell motility (B). SP600125 and Chir99021 treatments significantly inhibit cell migration (C, E), while etoposide shows no effect (D) in <t>U2OS</t> cells. Data are representative of three biological replicates; error bars represent standard deviation. Statistical significance was evaluated via Welch’s T test followed by Bonferroni Correction, * P
    U2os Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2052 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u2os cells/product/Millipore
    Average 99 stars, based on 2052 article reviews
    Price from $9.99 to $1999.99
    u2os cells - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Becton Dickinson u2os cells
    Schematic of the mechanism underlying the miR-192-5p/USP1 axis in 143B and <t>U2OS</t> cells. miR, microRNA; USP1, ubiquitin-specific protease 1.
    U2os Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 460 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u2os cells/product/Becton Dickinson
    Average 93 stars, based on 460 article reviews
    Price from $9.99 to $1999.99
    u2os cells - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    93
    MatTek u2os cells
    Mitotic spindle is chiral. a STED image (single z -plane) of metaphase spindle in a live HeLa cell expressing EGFP-CENP-A and EGFP-centrin1 (both shown in magenta) (left and middle; middle panel shows traces of microtubule bundles superimposed on the image), and in a live <t>U2OS</t> cell expressing CENP-A-GFP (magenta) (right). Microtubules are labeled with SiR-tubulin (green). b Imaging scheme of a vertically oriented spindle. c Imaging plane of a vertical spindle in a fixed HeLa cell expressing PRC1-GFP and mRFP-CENP-B (only PRC1-GFP is shown) (left); orthogonal plane of the same spindle (middle); arrows connecting starting and ending points of PRC1-GFP bundles traced upwards (right). Longer arrows roughly correspond to larger twist around the spindle axis (circle), colors show z -distance between starting and ending points, see color bar in g . d Schematic representation of the microtubule bundle helicity measurement. e Imaging scheme of a horizontally oriented spindle. f Horizontal spindle in a fixed HeLa cell expressing PRC1-GFP and mRFP-CENP-B, legend as in c . g Horizontal spindle in a fixed unlabeled HeLa cell immunostained for PRC1, with DNA stained by DAPI, legend as in c . Images in c left, and f , g middle are single planes; images in c middle, and f , g left are maximum intensity projections of five central planes. h Spindle helicity averaged over bundles for different conditions (vertical and horizontal spindles, fixed and live cells) and cell lines as indicated. Cell lines used were: HeLa cells expressing PRC1-GFP (1st, 2nd, 4th, and 5th bars), unlabeled HeLa cells immunostained for PRC1 (3rd bar), unlabeled U2OS cells immunostained for PRC1 (6th bar), U2OS cells expressing CENP-A-GFP, mCherry-α-tubulin, and photoactivatable (PA)-GFP-tubulin (7th bar). Data are representative of 4 independent experiments for unlabeled HeLa and U2OS cells immunostained for PRC1 and 3 independent experiments for all other conditions. Numbers represent the number of cells (top) and bundles (bottom). Data for individual cells are shown in Supplementary Fig. 1e . i Paper model of the spindle showing left-handed helicity of microtubule bundles and chirality of the whole spindle. Scale bars, 1 μm; error bars, s.e.m
    U2os Cells, supplied by MatTek, used in various techniques. Bioz Stars score: 93/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u2os cells/product/MatTek
    Average 93 stars, based on 358 article reviews
    Price from $9.99 to $1999.99
    u2os cells - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    94
    Thermo Fisher u2os cells
    Annexin V staining and TUNEL analysis in osteosarcoma cells treated with MTZ. (A) Annexin V staining results of <t>U2OS</t> and MG63 cells treated with MTZ (0, 0.5 and 1 µM) for 2 h. The representative images of the three assays are shown. The percentage of living, necrotic, early and late apoptotic cells are presented in each box of the graph. Graphs with quantitative data were based on the relative ratio of early, late, necrotic and live cells. The error bar indicates standard error. *P
    U2os Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 6715 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u2os cells/product/Thermo Fisher
    Average 94 stars, based on 6715 article reviews
    Price from $9.99 to $1999.99
    u2os cells - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    92
    Corning Life Sciences u2os cells
    A3H haplotype I has greater nuclear localization than haplotype II. ( a ) Representative images of A3H-I (untagged), A3H-II (untagged), A3B-HA and A3G-HA in SK-BR-3, HeLa and <t>U2OS</t> cells. The 20 μm scale applies to all images. ( b ) Whisker plots quantifying the subcellular localization data as nuclear-to-cytoplasmic ratios for n > 50 cells per condition. The average is shown, the error box represents the first and third quartiles, and the whiskers extend to the highest value within 1.5 × the interquartile range ( P values determined by two-tailed Welch's t -test).
    U2os Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u2os cells/product/Corning Life Sciences
    Average 92 stars, based on 149 article reviews
    Price from $9.99 to $1999.99
    u2os cells - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    93
    Greiner Bio u2os cells
    DAMP release from tumor cells treated with DTT-205 and DTT-304. The exposure of calreticulin (CALR) in human osteosarcoma <t>U2OS</t> cells was measured by flow cytometry using polyclonal anti-CALR antibody while excluding cells that lost cytoplasmic membrane integrity and thus incorporated the exclusion dye propidium iodide (PI) ( a, b ). Exodus of high mobility group box 1 (HMGB1) from the cells into cell culture supernatants was monitored by HMGB1-specific enzyme-linked immunosorbent assay ELISA. Absorbance was measured and concentrations were calculated based on standards ( c, d ). Both CALR and HMGB1 were emitted by both DTT-205 and DTT-304 in a dose-dependent fashion. The production of type I interferon (IFN) was measured in by reverse transcription quantitative real time polymerase chain reaction qRT-qPCR from purified mRNA of cells treated with DTT compounds ( e, f ) (mean ± SD of triplicate assessments; Student’s t test, * p
    U2os Cells, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 93/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u2os cells/product/Greiner Bio
    Average 93 stars, based on 190 article reviews
    Price from $9.99 to $1999.99
    u2os cells - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher tango tacr1 bla u2os cells
    DAMP release from tumor cells treated with DTT-205 and DTT-304. The exposure of calreticulin (CALR) in human osteosarcoma <t>U2OS</t> cells was measured by flow cytometry using polyclonal anti-CALR antibody while excluding cells that lost cytoplasmic membrane integrity and thus incorporated the exclusion dye propidium iodide (PI) ( a, b ). Exodus of high mobility group box 1 (HMGB1) from the cells into cell culture supernatants was monitored by HMGB1-specific enzyme-linked immunosorbent assay ELISA. Absorbance was measured and concentrations were calculated based on standards ( c, d ). Both CALR and HMGB1 were emitted by both DTT-205 and DTT-304 in a dose-dependent fashion. The production of type I interferon (IFN) was measured in by reverse transcription quantitative real time polymerase chain reaction qRT-qPCR from purified mRNA of cells treated with DTT compounds ( e, f ) (mean ± SD of triplicate assessments; Student’s t test, * p
    Tango Tacr1 Bla U2os Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tango tacr1 bla u2os cells/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    tango tacr1 bla u2os cells - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    92
    ibidi u2os cells
    Effects of CX-4945 and TDB on clone formation and survival. 100 <t>U2OS</t> cells were plated for each well, treated for 24 h with the indicated inhibitors, and then grown for further 6 days in the absence of the inhibitors. Two representative images are shown for each condition (2.5x objective, magnification-changer 1.6); at least two separate experiments in triplicate were performed.
    U2os Cells, supplied by ibidi, used in various techniques. Bioz Stars score: 92/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u2os cells/product/ibidi
    Average 92 stars, based on 105 article reviews
    Price from $9.99 to $1999.99
    u2os cells - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    RT53 induces unregulated necrotic cell death. (A) Ultrastructural analysis of RT53-mediated cell death. U2OS cells were left untreated (Control) or exposed to 15 μM of RT53 for 30 min. Cells were then analyzed by transmission electron microscopy following osmium tetroxide staining. (B) U2OS cells were exposed to 20 μM of RT53 in the presence or absence of 50 μM zVAD-fmk, 50 μM Necrostatin-1 (Nec-1) or 100 mM cyclosporin A (CsA) for 1 h. Necrotic cell death was monitored by lactate dehydrogenase (LDH) release from cells into the culture medium. The obtained values were normalized to those of the maximum LDH released (completely lysed) control. Data are means±s.e.m. ( n = 3).

    Journal: PLoS ONE

    Article Title: The anticancer peptide RT53 induces immunogenic cell death

    doi: 10.1371/journal.pone.0201220

    Figure Lengend Snippet: RT53 induces unregulated necrotic cell death. (A) Ultrastructural analysis of RT53-mediated cell death. U2OS cells were left untreated (Control) or exposed to 15 μM of RT53 for 30 min. Cells were then analyzed by transmission electron microscopy following osmium tetroxide staining. (B) U2OS cells were exposed to 20 μM of RT53 in the presence or absence of 50 μM zVAD-fmk, 50 μM Necrostatin-1 (Nec-1) or 100 mM cyclosporin A (CsA) for 1 h. Necrotic cell death was monitored by lactate dehydrogenase (LDH) release from cells into the culture medium. The obtained values were normalized to those of the maximum LDH released (completely lysed) control. Data are means±s.e.m. ( n = 3).

    Article Snippet: MRC-5 and U2OS cells were provided by Drs M. Dutreix and R. Fåhraeus, and were purchased from ATCC.

    Techniques: Transmission Assay, Electron Microscopy, Staining

    RT53 triggers calreticulin exposure as well as the release of HMGB1 and ATP. (A) U2OS cells stably expressing CRT-GFP were treated with 10 μM of RT53 or 1 μM mitoxantrone for 6 h, in the presence or absence of zVAD-fmk. Cells were then, fixed, stained for DNA, and examined by fluorescence microscopy. (B) U2OS cells were left untreated or treated with 10 μM of RT53 or 200 nM thapsigargin (TG) as a positive control for 6 h. Cell lysates were analyzed by Western blot for phosphorylated and total protein eIF2α. (C) U2OS cells were left untreated or exposed to increasing concentrations of RT53 for 3h. Extracellular HMGB1 was then measured in the culture supernatant. Data are means±s.e.m. ( n = 3). (D) U2OS cells were left untreated or exposed to increasing concentrations of RT53 for 3h. Extracellular ATP was then measured in the culture supernatant. Data are means±s.e.m. ( n = 3).

    Journal: PLoS ONE

    Article Title: The anticancer peptide RT53 induces immunogenic cell death

    doi: 10.1371/journal.pone.0201220

    Figure Lengend Snippet: RT53 triggers calreticulin exposure as well as the release of HMGB1 and ATP. (A) U2OS cells stably expressing CRT-GFP were treated with 10 μM of RT53 or 1 μM mitoxantrone for 6 h, in the presence or absence of zVAD-fmk. Cells were then, fixed, stained for DNA, and examined by fluorescence microscopy. (B) U2OS cells were left untreated or treated with 10 μM of RT53 or 200 nM thapsigargin (TG) as a positive control for 6 h. Cell lysates were analyzed by Western blot for phosphorylated and total protein eIF2α. (C) U2OS cells were left untreated or exposed to increasing concentrations of RT53 for 3h. Extracellular HMGB1 was then measured in the culture supernatant. Data are means±s.e.m. ( n = 3). (D) U2OS cells were left untreated or exposed to increasing concentrations of RT53 for 3h. Extracellular ATP was then measured in the culture supernatant. Data are means±s.e.m. ( n = 3).

    Article Snippet: MRC-5 and U2OS cells were provided by Drs M. Dutreix and R. Fåhraeus, and were purchased from ATCC.

    Techniques: Stable Transfection, Expressing, Staining, Fluorescence, Microscopy, Positive Control, Western Blot

    RT53 induces rapid cancer cells membranolysis. (A) Non-malignant (HaCat and MRC-5) and cancerous (U2OS, Lu1205, MCA205, Jurkat, HUT78 and HL-60) cells were treated with 20 μM of RT53. Plasma membrane permeability was evaluated at the designed time points by measuring extracellular LDH into the culture medium. The obtained values were normalized to those of the maximum LDH released (completely lysed) control. Data are means±s.e.m. ( n = 3) (B) U2OS cells were treated with 20 μM of RT53 or the control peptide RT53M for 3 h. Plasma membrane permeability was evaluated as in (A). (C) Mice red blood cells were incubated with 20 μM of RT53. Released hemoglobin was detected by densitometry at 540 nm. Hemoglobin release by cells treated with 1% Triton X-100 was used as 100% lysis control.

    Journal: PLoS ONE

    Article Title: The anticancer peptide RT53 induces immunogenic cell death

    doi: 10.1371/journal.pone.0201220

    Figure Lengend Snippet: RT53 induces rapid cancer cells membranolysis. (A) Non-malignant (HaCat and MRC-5) and cancerous (U2OS, Lu1205, MCA205, Jurkat, HUT78 and HL-60) cells were treated with 20 μM of RT53. Plasma membrane permeability was evaluated at the designed time points by measuring extracellular LDH into the culture medium. The obtained values were normalized to those of the maximum LDH released (completely lysed) control. Data are means±s.e.m. ( n = 3) (B) U2OS cells were treated with 20 μM of RT53 or the control peptide RT53M for 3 h. Plasma membrane permeability was evaluated as in (A). (C) Mice red blood cells were incubated with 20 μM of RT53. Released hemoglobin was detected by densitometry at 540 nm. Hemoglobin release by cells treated with 1% Triton X-100 was used as 100% lysis control.

    Article Snippet: MRC-5 and U2OS cells were provided by Drs M. Dutreix and R. Fåhraeus, and were purchased from ATCC.

    Techniques: Permeability, Mouse Assay, Incubation, Lysis

    Stable replication of the HPV18 genome in the U2OS cell line.

    Journal: PLoS ONE

    Article Title: The Cell Cycle Timing of Human Papillomavirus DNA Replication

    doi: 10.1371/journal.pone.0131675

    Figure Lengend Snippet: Stable replication of the HPV18 genome in the U2OS cell line.

    Article Snippet: Cell lines and transfection The U2OS cell line (obtained from American Type Culture Collection; ATCC no: HTB-96), which was used in all experiments, was grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal bovine serum.

    Techniques:

    Enriched OSIC activities correlate with accelerated sphere formation and expression of stem/progenitor cell-associated genes ( A ) Microscopy analysis of spheres cultivated in suspension for 12 days. Representative phase-contrast micrographs of spheres were shown with 400x magnification. UT1 or UT2 vs. U2OS: *P

    Journal: Oncogene

    Article Title: Human osteosarcoma CD49f−CD133+ cells: impaired in osteogenic fate while gain of tumorigenicity

    doi: 10.1038/onc.2012.438

    Figure Lengend Snippet: Enriched OSIC activities correlate with accelerated sphere formation and expression of stem/progenitor cell-associated genes ( A ) Microscopy analysis of spheres cultivated in suspension for 12 days. Representative phase-contrast micrographs of spheres were shown with 400x magnification. UT1 or UT2 vs. U2OS: *P

    Article Snippet: Human osteosarcoma cell lines U2OS, MG63, SAOS-2 were from ATCC (Manassas, VA, USA) and cultured as described ( ).

    Techniques: Expressing, Microscopy

    Enhanced OSIC self-renewal and tumorigenicity induce a progressive expansion of CD49f + cells forming the osteosarcoma mass ( A-C ) Expression levels of CD59, CD28, CD43, CD117, CD133 and STRO-1 (panel A) as well as CD49f (panels B, C) were determined by FACS in U2OS, UT2 and UT3 cells. ( D ) FACS analysis of CD49f expression in normal (MSC), KHOS/NP, and 8 different primary osteosarcoma cells. ( E F ) Inverse relationship between CD49f levels vs. time for tumor formation in serially transplanted (panel E) and the first transplanted cells (panel F).

    Journal: Oncogene

    Article Title: Human osteosarcoma CD49f−CD133+ cells: impaired in osteogenic fate while gain of tumorigenicity

    doi: 10.1038/onc.2012.438

    Figure Lengend Snippet: Enhanced OSIC self-renewal and tumorigenicity induce a progressive expansion of CD49f + cells forming the osteosarcoma mass ( A-C ) Expression levels of CD59, CD28, CD43, CD117, CD133 and STRO-1 (panel A) as well as CD49f (panels B, C) were determined by FACS in U2OS, UT2 and UT3 cells. ( D ) FACS analysis of CD49f expression in normal (MSC), KHOS/NP, and 8 different primary osteosarcoma cells. ( E F ) Inverse relationship between CD49f levels vs. time for tumor formation in serially transplanted (panel E) and the first transplanted cells (panel F).

    Article Snippet: Human osteosarcoma cell lines U2OS, MG63, SAOS-2 were from ATCC (Manassas, VA, USA) and cultured as described ( ).

    Techniques: Expressing, FACS

    Strong tumorigenicity of CD49f − CD133 + cells correlates with inhibited osteogenic capacity ( A ) Both CD133 + and CD133 − subpopulations of UT2 (left panel) or KHOS/PN cells (right panel) displayed osteogenic and adipogenic differentiation, as judged by ARS and ORO staining. ( B ) CD49f − CD133 + cells but not CD49f − CD133 − cells formed tumors in NOD/SCID mice (left panel); HE staining of CD49f − CD133 + xenograft cells showed the histological features of U2OS cells with osteoid (extra-cellular matrix) formation, as reflected by spindle cells with a vesicular nucleus often containing prominent nucleoli, frequent mitotic activities, and focal areas of osteoid formation (middle panel); engrafted CD49f − CD133 + cells differentiated to CD49f + cells in mice (right panel). ( C ) While both CD49f − CD133 + and CD49f − CD133 − cells showed a similar capacity for fat differentiation (sections 3 and 4), CD49f − CD133 + cells showed a reduced potential for bone differentiation, compared to CD49f − CD133 − cells and MSC (sections 1 vs. 2 and 6). ( D ) Microarray analysis revealed a decreased expression of osteogenic marker genes in UT2 cells. ( E F ) qRT-PCR analysis of osteogenic (panel E) and adipogenic marker gene expressions (panel F) in CD49f − and CD49f + subpopulations.

    Journal: Oncogene

    Article Title: Human osteosarcoma CD49f−CD133+ cells: impaired in osteogenic fate while gain of tumorigenicity

    doi: 10.1038/onc.2012.438

    Figure Lengend Snippet: Strong tumorigenicity of CD49f − CD133 + cells correlates with inhibited osteogenic capacity ( A ) Both CD133 + and CD133 − subpopulations of UT2 (left panel) or KHOS/PN cells (right panel) displayed osteogenic and adipogenic differentiation, as judged by ARS and ORO staining. ( B ) CD49f − CD133 + cells but not CD49f − CD133 − cells formed tumors in NOD/SCID mice (left panel); HE staining of CD49f − CD133 + xenograft cells showed the histological features of U2OS cells with osteoid (extra-cellular matrix) formation, as reflected by spindle cells with a vesicular nucleus often containing prominent nucleoli, frequent mitotic activities, and focal areas of osteoid formation (middle panel); engrafted CD49f − CD133 + cells differentiated to CD49f + cells in mice (right panel). ( C ) While both CD49f − CD133 + and CD49f − CD133 − cells showed a similar capacity for fat differentiation (sections 3 and 4), CD49f − CD133 + cells showed a reduced potential for bone differentiation, compared to CD49f − CD133 − cells and MSC (sections 1 vs. 2 and 6). ( D ) Microarray analysis revealed a decreased expression of osteogenic marker genes in UT2 cells. ( E F ) qRT-PCR analysis of osteogenic (panel E) and adipogenic marker gene expressions (panel F) in CD49f − and CD49f + subpopulations.

    Article Snippet: Human osteosarcoma cell lines U2OS, MG63, SAOS-2 were from ATCC (Manassas, VA, USA) and cultured as described ( ).

    Techniques: Staining, Mouse Assay, Microarray, Expressing, Marker, Quantitative RT-PCR

    Serial xenotransplantation enriches self-renewal and tumorigenicity of osteosarcoma cells ( A ) Self-renewal and tumorigenicity were assessed by the amount of cells injected and the time of tumor formation. Cells from the first U2OS xenograft were re-transplanted to yield the 1 st , 2 nd and 3 rd generations of U2OS cells (designated as UT1, UT2, and UT3). ( B ) HE staining determined that UT2 cells retained histological features of U2OS cells, as reflected by cellular polymorphism, nuclear hyperchromasia, and mitotic activities, together with osteoid formation that recapitulated the hallmark histological phenotype of the parental osteosarcoma. ( C ) UT2 cells remained capable of tumorigenicity in the bone. Tumor formation occurred in the right legs of mice with injection of UT2 cells (section ii). ( D ) HE staining of osteosarcoma xenograft formed in the femurs of the right legs (panel C), focally breaching the cortex, and extending into soft tissue (arrow and inset).

    Journal: Oncogene

    Article Title: Human osteosarcoma CD49f−CD133+ cells: impaired in osteogenic fate while gain of tumorigenicity

    doi: 10.1038/onc.2012.438

    Figure Lengend Snippet: Serial xenotransplantation enriches self-renewal and tumorigenicity of osteosarcoma cells ( A ) Self-renewal and tumorigenicity were assessed by the amount of cells injected and the time of tumor formation. Cells from the first U2OS xenograft were re-transplanted to yield the 1 st , 2 nd and 3 rd generations of U2OS cells (designated as UT1, UT2, and UT3). ( B ) HE staining determined that UT2 cells retained histological features of U2OS cells, as reflected by cellular polymorphism, nuclear hyperchromasia, and mitotic activities, together with osteoid formation that recapitulated the hallmark histological phenotype of the parental osteosarcoma. ( C ) UT2 cells remained capable of tumorigenicity in the bone. Tumor formation occurred in the right legs of mice with injection of UT2 cells (section ii). ( D ) HE staining of osteosarcoma xenograft formed in the femurs of the right legs (panel C), focally breaching the cortex, and extending into soft tissue (arrow and inset).

    Article Snippet: Human osteosarcoma cell lines U2OS, MG63, SAOS-2 were from ATCC (Manassas, VA, USA) and cultured as described ( ).

    Techniques: Injection, Staining, Mouse Assay

    PML-RARA t(15;17) mutation disrupts the ability of PML to bind TET2. A, Schematic showing primary structure of PML isoform I–VI. B, Co-IP analysis of TET2 and PML isoforms. pCI-Neo HA-TET2 and pCI-Neo Flag-PML isoforms (PML I-VI) were transfected into HEK293 cells. Then TET2 and PML I-VI were immunoprecipitated using anti-Flag and anti-HA antibodies. C, Fluorescent microscopy images of U2OS cells transfected with pCI-Neo HA-TET2 and pCI-Neo Flag-PML I-VI isoforms. These cells were stained with anti-HA and anti-PML antibodies. White arrows indicate the PML V shows no colocalizaiotn with TET2. Scale bar = 10 μm. D, Co-IP analysis of DNMT3A, TET2 and PML-RARA. HEK293 cells were transfected with pEGFP-DNMT3A, pEGFP-TET2 and pCI-Neo Flag-PML-RARA. Then GFP-DNMT3A, GFP-TET2 and Flag-PML-RARA were immunoprecipitated as noted. E, The 5hmC dot blot analysis of HEK293 transfected with pCI-NEO, pCI-NEO-Flag PML-RARA or pCI-NEO-Flag PML IV for 36 hrs. F, The 5hmC levels of HEK293 and U2OS transfected with pCI-NEO, pCI-NEO-Flag PML-RARA or pCI-NEO-Flag PML IV for 36 hrs. G, The 5hmC dot blot analysis of NB4 cells treated with 5 μM RA or 1 μM arsenic acid (As 2 O 3 ) for 36 hrs. H, The 5hmC levels of NB4 cells treated with RA or As 2 O 3 for 24 or 48 hrs. I, ). 1348 out of 2054 cases carried at least one mutation. Odd Ratio was calculated using the formula: Odd Ratio = (A * D)/(B * C), where A = number of cases altered in both genes; B = number of cases altered in G1 but not G2; C = number of cases altered in G2 but not G1; and D = number of cases altered in neither gene. Fisher’s exact test was used for the calculation of p value.

    Journal: Cancer research

    Article Title: PML recruits TET2 to regulate DNA modification and cell proliferation in response to chemotherapeutic agent

    doi: 10.1158/0008-5472.CAN-17-3091

    Figure Lengend Snippet: PML-RARA t(15;17) mutation disrupts the ability of PML to bind TET2. A, Schematic showing primary structure of PML isoform I–VI. B, Co-IP analysis of TET2 and PML isoforms. pCI-Neo HA-TET2 and pCI-Neo Flag-PML isoforms (PML I-VI) were transfected into HEK293 cells. Then TET2 and PML I-VI were immunoprecipitated using anti-Flag and anti-HA antibodies. C, Fluorescent microscopy images of U2OS cells transfected with pCI-Neo HA-TET2 and pCI-Neo Flag-PML I-VI isoforms. These cells were stained with anti-HA and anti-PML antibodies. White arrows indicate the PML V shows no colocalizaiotn with TET2. Scale bar = 10 μm. D, Co-IP analysis of DNMT3A, TET2 and PML-RARA. HEK293 cells were transfected with pEGFP-DNMT3A, pEGFP-TET2 and pCI-Neo Flag-PML-RARA. Then GFP-DNMT3A, GFP-TET2 and Flag-PML-RARA were immunoprecipitated as noted. E, The 5hmC dot blot analysis of HEK293 transfected with pCI-NEO, pCI-NEO-Flag PML-RARA or pCI-NEO-Flag PML IV for 36 hrs. F, The 5hmC levels of HEK293 and U2OS transfected with pCI-NEO, pCI-NEO-Flag PML-RARA or pCI-NEO-Flag PML IV for 36 hrs. G, The 5hmC dot blot analysis of NB4 cells treated with 5 μM RA or 1 μM arsenic acid (As 2 O 3 ) for 36 hrs. H, The 5hmC levels of NB4 cells treated with RA or As 2 O 3 for 24 or 48 hrs. I, ). 1348 out of 2054 cases carried at least one mutation. Odd Ratio was calculated using the formula: Odd Ratio = (A * D)/(B * C), where A = number of cases altered in both genes; B = number of cases altered in G1 but not G2; C = number of cases altered in G2 but not G1; and D = number of cases altered in neither gene. Fisher’s exact test was used for the calculation of p value.

    Article Snippet: HEK293 (human embryonic kidney), SCC-15 (human head and neck squamous cell carcinoma), SCC-25 (human head and neck squamous cell carcinoma), and U2OS (human osteosarcoma) cells from the ATCC were maintained in DMEM containing 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin, and streptomycin at 37°C under a humidified atmosphere of 5% CO2 .

    Techniques: Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Microscopy, Staining, Dot Blot

    The regions of TET2 to interact with PML. A, Schematic showing TET2-deletion mutants. B, Co-IP analysis of PML and TET2-deletion mutants. pS-Flag-SBP TET2-deletion mutants (F1-F4) or HA-PML were transfected into HEK293 cells. Then Flag tagged TET2-deletion mutants and HA-PML were immunoprecipitated. C, Fluorescent microscopy images of U2OS cells transfected with pS-Flag-SBP TET2-deletion mutants (F1-F4). Scale bar = 50 μm. D, The enlarged fluorescent microscopy images of the white rectangles in ( C ). E, Fluorescent microscopy images of U2OS cells transfected with pS-Flag-SBP TET2-deletion mutants (F1-F4) and HA-PML. Scale bar = 50 μm. F, The enlarged fluorescent microscopy images of the white rectangles in ( E ). White arrows indicate the colocalization between TET2-deletion mutants and PML.

    Journal: Cancer research

    Article Title: PML recruits TET2 to regulate DNA modification and cell proliferation in response to chemotherapeutic agent

    doi: 10.1158/0008-5472.CAN-17-3091

    Figure Lengend Snippet: The regions of TET2 to interact with PML. A, Schematic showing TET2-deletion mutants. B, Co-IP analysis of PML and TET2-deletion mutants. pS-Flag-SBP TET2-deletion mutants (F1-F4) or HA-PML were transfected into HEK293 cells. Then Flag tagged TET2-deletion mutants and HA-PML were immunoprecipitated. C, Fluorescent microscopy images of U2OS cells transfected with pS-Flag-SBP TET2-deletion mutants (F1-F4). Scale bar = 50 μm. D, The enlarged fluorescent microscopy images of the white rectangles in ( C ). E, Fluorescent microscopy images of U2OS cells transfected with pS-Flag-SBP TET2-deletion mutants (F1-F4) and HA-PML. Scale bar = 50 μm. F, The enlarged fluorescent microscopy images of the white rectangles in ( E ). White arrows indicate the colocalization between TET2-deletion mutants and PML.

    Article Snippet: HEK293 (human embryonic kidney), SCC-15 (human head and neck squamous cell carcinoma), SCC-25 (human head and neck squamous cell carcinoma), and U2OS (human osteosarcoma) cells from the ATCC were maintained in DMEM containing 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin, and streptomycin at 37°C under a humidified atmosphere of 5% CO2 .

    Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Microscopy

    PML physically binds to TET2 and recruits TET2 to PML nuclear bodies. A, Schemamtic showing SILAC-labeling and mass spectrometry analysis of anti-TET2 co-IP. B, The 5mC levels (5mC/C) of HEK293 cells transfected with the plasmids encoding 65 potential functional partners of TET2. The pCDH lentivector was used as control and the 5hmC level of pCDH-transfected HEK293 was taken as 1. C, Co-IP analysis of endogenous TET2 and PML in HEK293 cells. D, Co-IP analysis of EGFP-tagged TET2 and HA-tagged PML. pEGFP-DNMT3A, pEGFP-TET2 or HA-PML (IV) were transfected in HEK293 cells. Then GFP-DNMT3A, GFP-TET2 and HA-PML were immunoprecipitated as noted. E, Fluorescent microscopy images of U2OS cells transfected with expression vectors containing pEGFP-TET2, pEGFP-DNMT3A or pEGFP-DNMT3B. Scale bar = 50 μm. F, The enlarged fluorescent microscopy images of the white rectangles in ( E ). G, Fluorescent microscopy images of U2OS cells transfected with HA-PML, pCI-Neo Flag-TET2, pEGFP-DNMT3A or pEGFP-DNMT3B. These cells were stained with anti-Flag (red, first-column panels) and anti-PML (blue, third-column panels) antibodies. Scale bar = 50 μm. H, The enlarged fluorescent microscopy images of the white rectangles in G . White arrows indicate that only Flag-TET2 is colocalized with PML-NBs.

    Journal: Cancer research

    Article Title: PML recruits TET2 to regulate DNA modification and cell proliferation in response to chemotherapeutic agent

    doi: 10.1158/0008-5472.CAN-17-3091

    Figure Lengend Snippet: PML physically binds to TET2 and recruits TET2 to PML nuclear bodies. A, Schemamtic showing SILAC-labeling and mass spectrometry analysis of anti-TET2 co-IP. B, The 5mC levels (5mC/C) of HEK293 cells transfected with the plasmids encoding 65 potential functional partners of TET2. The pCDH lentivector was used as control and the 5hmC level of pCDH-transfected HEK293 was taken as 1. C, Co-IP analysis of endogenous TET2 and PML in HEK293 cells. D, Co-IP analysis of EGFP-tagged TET2 and HA-tagged PML. pEGFP-DNMT3A, pEGFP-TET2 or HA-PML (IV) were transfected in HEK293 cells. Then GFP-DNMT3A, GFP-TET2 and HA-PML were immunoprecipitated as noted. E, Fluorescent microscopy images of U2OS cells transfected with expression vectors containing pEGFP-TET2, pEGFP-DNMT3A or pEGFP-DNMT3B. Scale bar = 50 μm. F, The enlarged fluorescent microscopy images of the white rectangles in ( E ). G, Fluorescent microscopy images of U2OS cells transfected with HA-PML, pCI-Neo Flag-TET2, pEGFP-DNMT3A or pEGFP-DNMT3B. These cells were stained with anti-Flag (red, first-column panels) and anti-PML (blue, third-column panels) antibodies. Scale bar = 50 μm. H, The enlarged fluorescent microscopy images of the white rectangles in G . White arrows indicate that only Flag-TET2 is colocalized with PML-NBs.

    Article Snippet: HEK293 (human embryonic kidney), SCC-15 (human head and neck squamous cell carcinoma), SCC-25 (human head and neck squamous cell carcinoma), and U2OS (human osteosarcoma) cells from the ATCC were maintained in DMEM containing 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin, and streptomycin at 37°C under a humidified atmosphere of 5% CO2 .

    Techniques: Labeling, Mass Spectrometry, Co-Immunoprecipitation Assay, Transfection, Functional Assay, Immunoprecipitation, Microscopy, Expressing, Staining

    Acute perturbation of dynamin with dynasore influences α-actinin distribution but not the overall F-actin organization. U2-OS cells were treated with 80 μ m dynasore or DMSO (0.4%) for 20 min at 37 °C before fixing and immunostaining

    Journal: The Journal of Biological Chemistry

    Article Title: Dynamin2 GTPase and Cortactin Remodel Actin Filaments *

    doi: 10.1074/jbc.M109.024398

    Figure Lengend Snippet: Acute perturbation of dynamin with dynasore influences α-actinin distribution but not the overall F-actin organization. U2-OS cells were treated with 80 μ m dynasore or DMSO (0.4%) for 20 min at 37 °C before fixing and immunostaining

    Article Snippet: Dynamin1, which is expressed at low amounts by U2-OS cells, was unaltered in cells treated with siRNAs targeting dynamin2 ( A ).

    Techniques: Immunostaining

    Dynamin2 influences the distributions of F-actin and non-muscle myosin IIA; exogenous rat dynamin2 restores the myosin IIA distribution in U2-OS cells. A , cells were fixed and stained with rhodamine-phalloidin ( left panels ) or anti-myosin IIA ( right panels

    Journal: The Journal of Biological Chemistry

    Article Title: Dynamin2 GTPase and Cortactin Remodel Actin Filaments *

    doi: 10.1074/jbc.M109.024398

    Figure Lengend Snippet: Dynamin2 influences the distributions of F-actin and non-muscle myosin IIA; exogenous rat dynamin2 restores the myosin IIA distribution in U2-OS cells. A , cells were fixed and stained with rhodamine-phalloidin ( left panels ) or anti-myosin IIA ( right panels

    Article Snippet: Dynamin1, which is expressed at low amounts by U2-OS cells, was unaltered in cells treated with siRNAs targeting dynamin2 ( A ).

    Techniques: Staining

    Dynamin2 influences the distributions of F-actin and α-actinin in U2-OS cells. A , dynamin2 was efficiently depleted from U2-OS cells using two different siRNAs targeting human dynamin2. Cell extracts obtained 48–72 h after treatment with

    Journal: The Journal of Biological Chemistry

    Article Title: Dynamin2 GTPase and Cortactin Remodel Actin Filaments *

    doi: 10.1074/jbc.M109.024398

    Figure Lengend Snippet: Dynamin2 influences the distributions of F-actin and α-actinin in U2-OS cells. A , dynamin2 was efficiently depleted from U2-OS cells using two different siRNAs targeting human dynamin2. Cell extracts obtained 48–72 h after treatment with

    Article Snippet: Dynamin1, which is expressed at low amounts by U2-OS cells, was unaltered in cells treated with siRNAs targeting dynamin2 ( A ).

    Techniques:

    Cardinal motif NRF1 dictates the recruitment of KDM LSD1 via TF NRF1. ( A ) Representative examples of ChIP-seq tracks showing precise co-alignment of NRF1 and LSD1. These particular loci ( ZWINT and LINS-ASB7 ) were selected as representative despite being rare examples in which NRF1 binds nearby NFYB, but they should help to emphasize the good co-alignment between LSD1 and NRF1 using NFYB as reference. The track of ChIP-seq data for H3K4me3 was also included as reference. Annotation of Ref-seq genes is included. ( B ) Meta-analysis of sequencing read density of LSD1 ChIP-seq (orange) and NRF1 ChIP-seq (red) signals around NRF1 peaks (center of the panel). ( C ) Left: Scheme of two constructs engineered to contain 3× wild-type NRF1 sequences (3xwtNRF1, as in Figure 2A ) or 3× mutated NRF1 sequences (3xmutNRF1) upstream the luciferase reporter gene. Panels: Luciferase assay with 3xwtNRF1- or 3xmutNRF1-transected U2OS cells (left panel) and ChIP analyses of NRF1 and LSD1 at 3xwtNRF1 and 3xmutNRF1 sites (middle and right panels, respectively). For ChIP analyses, two regions were amplified (labeled in the scheme, left): one region covering 3xwtNRF1 or 3xmutNRF1 sites (depending on the construct), amplified with primers named as ‘site’; and the other region covering a distal, control area (the same in both constructs), amplified with primers named as ‘ctl’. ( D ) Co-immunoprecipitation (IP) assay with the set of antibodies indicated on top of the panel, and detection with the set of antibodies indicated in the left of the panel. ( E ) Size exclusion chromatography (Superose 6) of nuclear extracts obtained from MCF7 cells. Analysis of elution fractions by Western blot using anti-LSD1 (top) and anti-NRF1 (bottom) antibodies. On top of the Western blot, elution of known molecular size markers is indicated (arrows). Voided volume was determined with Blue Dextran 2000 ( > 2MDa).

    Journal: PLoS Genetics

    Article Title: Decoding a Signature-Based Model of Transcription Cofactor Recruitment Dictated by Cardinal Cis-Regulatory Elements in Proximal Promoter Regions

    doi: 10.1371/journal.pgen.1003906

    Figure Lengend Snippet: Cardinal motif NRF1 dictates the recruitment of KDM LSD1 via TF NRF1. ( A ) Representative examples of ChIP-seq tracks showing precise co-alignment of NRF1 and LSD1. These particular loci ( ZWINT and LINS-ASB7 ) were selected as representative despite being rare examples in which NRF1 binds nearby NFYB, but they should help to emphasize the good co-alignment between LSD1 and NRF1 using NFYB as reference. The track of ChIP-seq data for H3K4me3 was also included as reference. Annotation of Ref-seq genes is included. ( B ) Meta-analysis of sequencing read density of LSD1 ChIP-seq (orange) and NRF1 ChIP-seq (red) signals around NRF1 peaks (center of the panel). ( C ) Left: Scheme of two constructs engineered to contain 3× wild-type NRF1 sequences (3xwtNRF1, as in Figure 2A ) or 3× mutated NRF1 sequences (3xmutNRF1) upstream the luciferase reporter gene. Panels: Luciferase assay with 3xwtNRF1- or 3xmutNRF1-transected U2OS cells (left panel) and ChIP analyses of NRF1 and LSD1 at 3xwtNRF1 and 3xmutNRF1 sites (middle and right panels, respectively). For ChIP analyses, two regions were amplified (labeled in the scheme, left): one region covering 3xwtNRF1 or 3xmutNRF1 sites (depending on the construct), amplified with primers named as ‘site’; and the other region covering a distal, control area (the same in both constructs), amplified with primers named as ‘ctl’. ( D ) Co-immunoprecipitation (IP) assay with the set of antibodies indicated on top of the panel, and detection with the set of antibodies indicated in the left of the panel. ( E ) Size exclusion chromatography (Superose 6) of nuclear extracts obtained from MCF7 cells. Analysis of elution fractions by Western blot using anti-LSD1 (top) and anti-NRF1 (bottom) antibodies. On top of the Western blot, elution of known molecular size markers is indicated (arrows). Voided volume was determined with Blue Dextran 2000 ( > 2MDa).

    Article Snippet: Human osteosarcoma U2OS cells, human embryonic kidney (HEK) 293T cells, and human neuroblastoma SH-SY5Y cells were cultured according to The American Type Culture Collection (ATCC) protocols.

    Techniques: Chromatin Immunoprecipitation, Sequencing, Construct, Luciferase, Amplification, Labeling, CTL Assay, Immunoprecipitation, Size-exclusion Chromatography, Western Blot

    Diversity of functional outcomes associated with NRF1/LSD1 recruitment at −150/+50 bp regions. ( A ) Left: Western blot analysis of NRF1 and LSD1 in whole cell extracts obtained from MCF7 cells in which NRF1 or LSD1 (indicated on top) were depleted by siRNA. Actin is shown as loading control. Scrambled (CTL) siRNA is included as control of transfection. Panels: RT-qPCR analysis of genes that have been identified in this study as having promoters co-occupied by NRF1 and LSD1 (NRF1 + and LSD1 + targets). Gene names are indicated on top of each panel. Treatments are indicated at the bottom. Scrambled (CTL) siRNA was used as control. The y-axis refers to normalized expression to levels of ACTB mRNA. ( B ) Top: Western blot analysis as shown in A (left panel) but in U2OS cells. Bottom: Venn diagram depicting the overlap of genes affected by NRF1 (light red circle) and LSD1 (orange circle) siRNA treatments with respect to control (CTL) siRNA in U2OS cells, based on microarray. The number of total and category-wise genes affected by the treatments are indicated, as well as the statistical significance of the overlap. ( C ) Matrix of motif enrichment of cardinal motifs in −150/+50 bp regions of genes identified by microarray as affected by NRF1 (left) or LSD1 (right) knockdown in U2OS cells. Enrichment levels were determined with respect to background frequencies of the same motifs in −150/+50 bp regions. Motif enrichments higher than background are shown as a gradient of blue, while motif enrichments lower than background are shown as a gradient of red. No motif enrichment is shown as white. The number of promoters analyzed is also indicated. ( D ) Venn diagram depicting the overlap of microarray-identified genes classified based on their type of response to NRF1 or LSD1 siRNA treatments. The numbers of genes in the overlaps are indicated, as well as the numbers of genes that did not overlap and the total numbers. ( E ) Combined analysis of microarray and ChIP-based data. We combined microarray results identifying NRF1 and/or LSD1 siRNA-mediated effects and ChIP-DSL data identifying NRF1 and/or LSD1 occupied promoters. Gene classes based on ( D ). The y-axis refers to the relative enrichment over background in the number of genes that were affected by both NRF1 and LSD1 siRNA treatments and that had promoters occupied by LSD1 (orange) and/or NRF1 (red; see text for more details). A ratio of ‘fold over background’ higher or lower than 1 ( > 1 or

    Journal: PLoS Genetics

    Article Title: Decoding a Signature-Based Model of Transcription Cofactor Recruitment Dictated by Cardinal Cis-Regulatory Elements in Proximal Promoter Regions

    doi: 10.1371/journal.pgen.1003906

    Figure Lengend Snippet: Diversity of functional outcomes associated with NRF1/LSD1 recruitment at −150/+50 bp regions. ( A ) Left: Western blot analysis of NRF1 and LSD1 in whole cell extracts obtained from MCF7 cells in which NRF1 or LSD1 (indicated on top) were depleted by siRNA. Actin is shown as loading control. Scrambled (CTL) siRNA is included as control of transfection. Panels: RT-qPCR analysis of genes that have been identified in this study as having promoters co-occupied by NRF1 and LSD1 (NRF1 + and LSD1 + targets). Gene names are indicated on top of each panel. Treatments are indicated at the bottom. Scrambled (CTL) siRNA was used as control. The y-axis refers to normalized expression to levels of ACTB mRNA. ( B ) Top: Western blot analysis as shown in A (left panel) but in U2OS cells. Bottom: Venn diagram depicting the overlap of genes affected by NRF1 (light red circle) and LSD1 (orange circle) siRNA treatments with respect to control (CTL) siRNA in U2OS cells, based on microarray. The number of total and category-wise genes affected by the treatments are indicated, as well as the statistical significance of the overlap. ( C ) Matrix of motif enrichment of cardinal motifs in −150/+50 bp regions of genes identified by microarray as affected by NRF1 (left) or LSD1 (right) knockdown in U2OS cells. Enrichment levels were determined with respect to background frequencies of the same motifs in −150/+50 bp regions. Motif enrichments higher than background are shown as a gradient of blue, while motif enrichments lower than background are shown as a gradient of red. No motif enrichment is shown as white. The number of promoters analyzed is also indicated. ( D ) Venn diagram depicting the overlap of microarray-identified genes classified based on their type of response to NRF1 or LSD1 siRNA treatments. The numbers of genes in the overlaps are indicated, as well as the numbers of genes that did not overlap and the total numbers. ( E ) Combined analysis of microarray and ChIP-based data. We combined microarray results identifying NRF1 and/or LSD1 siRNA-mediated effects and ChIP-DSL data identifying NRF1 and/or LSD1 occupied promoters. Gene classes based on ( D ). The y-axis refers to the relative enrichment over background in the number of genes that were affected by both NRF1 and LSD1 siRNA treatments and that had promoters occupied by LSD1 (orange) and/or NRF1 (red; see text for more details). A ratio of ‘fold over background’ higher or lower than 1 ( > 1 or

    Article Snippet: Human osteosarcoma U2OS cells, human embryonic kidney (HEK) 293T cells, and human neuroblastoma SH-SY5Y cells were cultured according to The American Type Culture Collection (ATCC) protocols.

    Techniques: Functional Assay, Western Blot, CTL Assay, Transfection, Quantitative RT-PCR, Expressing, Microarray, Chromatin Immunoprecipitation

    Primary screening strategy narrowed a library of 150 000 compounds down to 5786 hits. ( A ) Ribosome labeling strategy was validated in U2OS cells by pulse labeling with two distinct fluorescent HaloTag ligands with (TMR Red) and without (Oregon Green) a 3 h chase period prior to fixation and imaging. Images are displayed without false color. ( B ) Primary HTS screen strategy deployed the HaloTag technology for a fluorescent pulse labeling of newly synthesized ribosomes.

    Journal: The Biochemical journal

    Article Title: Discovery of novel inhibitors of ribosome biogenesis by innovative high throughput screening strategies

    doi: 10.1042/BCJ20190207

    Figure Lengend Snippet: Primary screening strategy narrowed a library of 150 000 compounds down to 5786 hits. ( A ) Ribosome labeling strategy was validated in U2OS cells by pulse labeling with two distinct fluorescent HaloTag ligands with (TMR Red) and without (Oregon Green) a 3 h chase period prior to fixation and imaging. Images are displayed without false color. ( B ) Primary HTS screen strategy deployed the HaloTag technology for a fluorescent pulse labeling of newly synthesized ribosomes.

    Article Snippet: Cell culture and compound treatment A375, malignant melanoma cells and U2OS osteosarcoma cells were obtained from ATCC.

    Techniques: Labeling, Imaging, Synthesized

    Rapid accumulation of p53 at sites of DNA damage. ( A ) Representative fluorescent micrographs showing the rapid recruitment of endogenous p53 with a CRISPR-KI neongreen tag in RPE1 cells. ( B ) Representative fluorescent micrographs showing rapid accumulation of wildtype (WT) p53-EGFP fusion protein following overexpression in U2OS cells. White dotted circle indicates irradiated area. The diameter of ROIs for quantification is set at 2.5 μm if not indicated otherwise, while the full width at half maxima of the UV laser beam at focal plane was approximately 850 nm. Colored dotted line indicates single exponential fitting. ( C ) The rapid accumulation is not detected with the p53 DNA-binding mutant, R248Q. ( D-E ) Quantification of fluorescence intensity within irradiated area or region of interest (ROI) of ( D ) endogenous p53-neongreen in RPE1 cells (mean±s.d., n=19) and ( E ) WT p53-EGFP in U2OS cells (mean±s.d., n=30). Signal intensity measurements outside the ROI indicate that p53 protein is being redistributed rapidly following irradiation. ( F ) Global and ( G ) zoom in view of immunostaining of endogenous p53 and γH2AX in U2OS cells 5 minutes after irradiation. Yellow line indicates the region used for intensity profile analysis. ( H ) Corresponding fluorescence intensity profile demonstrating co-localization of p53 and γH2AX at early time points after laser irradiation. ( I ) The staining of p53 at DNA damage sites is not detectable in U2OS treated with siRNA against p53 and the staining of γH2AX appears more diffusive.

    Journal: bioRxiv

    Article Title: Both DNA binding domains of p53 are required for its ultra-rapid recruitment to sites of UV damage

    doi: 10.1101/2020.02.09.938993

    Figure Lengend Snippet: Rapid accumulation of p53 at sites of DNA damage. ( A ) Representative fluorescent micrographs showing the rapid recruitment of endogenous p53 with a CRISPR-KI neongreen tag in RPE1 cells. ( B ) Representative fluorescent micrographs showing rapid accumulation of wildtype (WT) p53-EGFP fusion protein following overexpression in U2OS cells. White dotted circle indicates irradiated area. The diameter of ROIs for quantification is set at 2.5 μm if not indicated otherwise, while the full width at half maxima of the UV laser beam at focal plane was approximately 850 nm. Colored dotted line indicates single exponential fitting. ( C ) The rapid accumulation is not detected with the p53 DNA-binding mutant, R248Q. ( D-E ) Quantification of fluorescence intensity within irradiated area or region of interest (ROI) of ( D ) endogenous p53-neongreen in RPE1 cells (mean±s.d., n=19) and ( E ) WT p53-EGFP in U2OS cells (mean±s.d., n=30). Signal intensity measurements outside the ROI indicate that p53 protein is being redistributed rapidly following irradiation. ( F ) Global and ( G ) zoom in view of immunostaining of endogenous p53 and γH2AX in U2OS cells 5 minutes after irradiation. Yellow line indicates the region used for intensity profile analysis. ( H ) Corresponding fluorescence intensity profile demonstrating co-localization of p53 and γH2AX at early time points after laser irradiation. ( I ) The staining of p53 at DNA damage sites is not detectable in U2OS treated with siRNA against p53 and the staining of γH2AX appears more diffusive.

    Article Snippet: Cell culture U2OS and Saos2 (human osteosarcoma), SW-13 cells (human primary small cell carcinoma in adrenal gland/cortex), BxPC3 (pancreatic adenocarcinoma) and SK-BR-3 (mammary adenocarcinoma) were obtained from the American Type Culture Collection, ATCC, (catalogue no. HTB-96™, HTB-85™, CCL-105™, CRL-1687™ and HTB-30™ respectively).

    Techniques: CRISPR, Over Expression, Irradiation, Binding Assay, Mutagenesis, Fluorescence, Immunostaining, Staining

    Rapid accumulation of p53 is dependent on PARP activity. ( A ) Quantification of fluorescence intensity within ROI over 300 minutes of wildtype (WT) p53-EGFP and PARP-cb-RFP following irradiation. ( B-C ) Representative fluorescent micrographs showing recruitment of p53-EGFP and PARP-cb-RFP in the first 100 minutes following irradiation, in the absence and presence of PARP inhibition (PARPi), in U2OS cells. (D-E) Kymograph showing different p53 recruitment dynamics following the dotted yellow line showing in panel B C, respectively. ( F ) Rapid accumulation of WT p53-EGFP in U2OS cells is suppressed or delayed to different extents by various inhibitors (20nM ATRi, 50nM ATMi, 10nM PARPi) as measured within the first 40 seconds following irradiation. ( G ) Magnitude of accumulation of WT p53-EGFP as measured by fluorescence intensity within ROI in the presence of various inhibitors in U2OS cells. ( H, J, L ) Quantification of fluorescence intensity within ROI of WT p53-EGFP and PARP-cb-RFP following irradiation, in absence and presence of various inhibitors, over 5 hours in U2OS cells. ( I, K, M ) Corresponding zoomed in views of F, H, J, focusing on the first 40 minutes of WT p53-EGFP and PARP-cb-RFP recruitment, in absence and presence of various inhibitors, in U2OS cells. ( O ) Protein sequences of PBM4 and PBM10 mutants with disrupted p53-PARP interaction. ( P Q ) The lack of recruitment of both PBM4 and PBM10 mutants in comparison to the p53-WT in ( P ) U2OS and ( Q ) Saos2 respectively.

    Journal: bioRxiv

    Article Title: Both DNA binding domains of p53 are required for its ultra-rapid recruitment to sites of UV damage

    doi: 10.1101/2020.02.09.938993

    Figure Lengend Snippet: Rapid accumulation of p53 is dependent on PARP activity. ( A ) Quantification of fluorescence intensity within ROI over 300 minutes of wildtype (WT) p53-EGFP and PARP-cb-RFP following irradiation. ( B-C ) Representative fluorescent micrographs showing recruitment of p53-EGFP and PARP-cb-RFP in the first 100 minutes following irradiation, in the absence and presence of PARP inhibition (PARPi), in U2OS cells. (D-E) Kymograph showing different p53 recruitment dynamics following the dotted yellow line showing in panel B C, respectively. ( F ) Rapid accumulation of WT p53-EGFP in U2OS cells is suppressed or delayed to different extents by various inhibitors (20nM ATRi, 50nM ATMi, 10nM PARPi) as measured within the first 40 seconds following irradiation. ( G ) Magnitude of accumulation of WT p53-EGFP as measured by fluorescence intensity within ROI in the presence of various inhibitors in U2OS cells. ( H, J, L ) Quantification of fluorescence intensity within ROI of WT p53-EGFP and PARP-cb-RFP following irradiation, in absence and presence of various inhibitors, over 5 hours in U2OS cells. ( I, K, M ) Corresponding zoomed in views of F, H, J, focusing on the first 40 minutes of WT p53-EGFP and PARP-cb-RFP recruitment, in absence and presence of various inhibitors, in U2OS cells. ( O ) Protein sequences of PBM4 and PBM10 mutants with disrupted p53-PARP interaction. ( P Q ) The lack of recruitment of both PBM4 and PBM10 mutants in comparison to the p53-WT in ( P ) U2OS and ( Q ) Saos2 respectively.

    Article Snippet: Cell culture U2OS and Saos2 (human osteosarcoma), SW-13 cells (human primary small cell carcinoma in adrenal gland/cortex), BxPC3 (pancreatic adenocarcinoma) and SK-BR-3 (mammary adenocarcinoma) were obtained from the American Type Culture Collection, ATCC, (catalogue no. HTB-96™, HTB-85™, CCL-105™, CRL-1687™ and HTB-30™ respectively).

    Techniques: Activity Assay, Fluorescence, Irradiation, Inhibition

    Common p53 mutants in human cancer fail to be rapidly recruited to sites of DNA damage. ( A) Frequency analysis of missense and nonsense mutations of TP53 in human cancer based on data available on the COSMIC (Catalogue of Somatic Mutations in Cancer) database. Common ‘hotspot’ areas for mutations are R175, R248 and R273. ( B ) Diagram of human wildtype p53 with various domains indicated. ( C ) Magnitude of accumulation of wildtype (WT) and various mutant p53 as measured by fluorescence intensity within ROI in U2OS cells. (From left to right, n= 54, 18, 20, 18, 30, 21, 25, 26, 24, 19, 15, 22, 20, 25, 31, 30, 27, 25, 25, 25, 23, 21, 22, 23) ( D ) Comparison of the magnitude of accumulation of wildtype (WT) and various mutant p53s in U2OS and Saos2 cells following irradiation.

    Journal: bioRxiv

    Article Title: Both DNA binding domains of p53 are required for its ultra-rapid recruitment to sites of UV damage

    doi: 10.1101/2020.02.09.938993

    Figure Lengend Snippet: Common p53 mutants in human cancer fail to be rapidly recruited to sites of DNA damage. ( A) Frequency analysis of missense and nonsense mutations of TP53 in human cancer based on data available on the COSMIC (Catalogue of Somatic Mutations in Cancer) database. Common ‘hotspot’ areas for mutations are R175, R248 and R273. ( B ) Diagram of human wildtype p53 with various domains indicated. ( C ) Magnitude of accumulation of wildtype (WT) and various mutant p53 as measured by fluorescence intensity within ROI in U2OS cells. (From left to right, n= 54, 18, 20, 18, 30, 21, 25, 26, 24, 19, 15, 22, 20, 25, 31, 30, 27, 25, 25, 25, 23, 21, 22, 23) ( D ) Comparison of the magnitude of accumulation of wildtype (WT) and various mutant p53s in U2OS and Saos2 cells following irradiation.

    Article Snippet: Cell culture U2OS and Saos2 (human osteosarcoma), SW-13 cells (human primary small cell carcinoma in adrenal gland/cortex), BxPC3 (pancreatic adenocarcinoma) and SK-BR-3 (mammary adenocarcinoma) were obtained from the American Type Culture Collection, ATCC, (catalogue no. HTB-96™, HTB-85™, CCL-105™, CRL-1687™ and HTB-30™ respectively).

    Techniques: Mutagenesis, Fluorescence, Irradiation

    Prednisolone modulates RANKL and OPG expression in U2OS cells. A: Cells were incubated with prednisolone (10 −8 mol/L, 2 hours) and analysis of mRNA expression conducted as detailed in . Blot is representative of three experiments whose

    Journal: The American Journal of Pathology

    Article Title: Calcitonin and Prednisolone Display Antagonistic Actions on Bone and Have Synergistic Effects in Experimental Arthritis

    doi: 10.2353/ajpath.2007.060830

    Figure Lengend Snippet: Prednisolone modulates RANKL and OPG expression in U2OS cells. A: Cells were incubated with prednisolone (10 −8 mol/L, 2 hours) and analysis of mRNA expression conducted as detailed in . Blot is representative of three experiments whose

    Article Snippet: The osteosarcoma cell lines U2OS and Saos-2 and the breast cancer cell line T47D (all from the American Type Culture Collection catalog; distributed by LGC Promochem, Teddington, Middlesex, UK) were cultured in McCoy’s medium with 10% fetal calf serum (Gibco, Paisley, UK), 100 U/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO2 /95% air at 37°C and passaged two times a week.

    Techniques: Expressing, Incubation

    GC receptor binding assay in U2OS cells. A: Scatchard plot analysis with different concentrations of the tracer [ 3 H]dexamethasone indicates the existence of a single specific binding site in U2OS cells. The parameters calculated from this

    Journal: The American Journal of Pathology

    Article Title: Calcitonin and Prednisolone Display Antagonistic Actions on Bone and Have Synergistic Effects in Experimental Arthritis

    doi: 10.2353/ajpath.2007.060830

    Figure Lengend Snippet: GC receptor binding assay in U2OS cells. A: Scatchard plot analysis with different concentrations of the tracer [ 3 H]dexamethasone indicates the existence of a single specific binding site in U2OS cells. The parameters calculated from this

    Article Snippet: The osteosarcoma cell lines U2OS and Saos-2 and the breast cancer cell line T47D (all from the American Type Culture Collection catalog; distributed by LGC Promochem, Teddington, Middlesex, UK) were cultured in McCoy’s medium with 10% fetal calf serum (Gibco, Paisley, UK), 100 U/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO2 /95% air at 37°C and passaged two times a week.

    Techniques: Reporter Assay, Binding Assay

    CT modulates RANKL and OPG expression in U2OS cells. A: Cell incubation with CT (10 −10 mol/L, 2 hours) down-regulates RANKL mRNA expression and up-regulates OPG mRNA expression, as detected by RT-PCR. The representative blot shows the RANKL product

    Journal: The American Journal of Pathology

    Article Title: Calcitonin and Prednisolone Display Antagonistic Actions on Bone and Have Synergistic Effects in Experimental Arthritis

    doi: 10.2353/ajpath.2007.060830

    Figure Lengend Snippet: CT modulates RANKL and OPG expression in U2OS cells. A: Cell incubation with CT (10 −10 mol/L, 2 hours) down-regulates RANKL mRNA expression and up-regulates OPG mRNA expression, as detected by RT-PCR. The representative blot shows the RANKL product

    Article Snippet: The osteosarcoma cell lines U2OS and Saos-2 and the breast cancer cell line T47D (all from the American Type Culture Collection catalog; distributed by LGC Promochem, Teddington, Middlesex, UK) were cultured in McCoy’s medium with 10% fetal calf serum (Gibco, Paisley, UK), 100 U/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO2 /95% air at 37°C and passaged two times a week.

    Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction

    Expression of a functional calcitonin receptor in U2OS cells. A: Expression of the specific mRNA for the calcitonin receptor as detected by RT-PCR. U2OS cells show a positive band at the expected size (386 bp) ( lane 1 ), whereas another human osteosarcoma

    Journal: The American Journal of Pathology

    Article Title: Calcitonin and Prednisolone Display Antagonistic Actions on Bone and Have Synergistic Effects in Experimental Arthritis

    doi: 10.2353/ajpath.2007.060830

    Figure Lengend Snippet: Expression of a functional calcitonin receptor in U2OS cells. A: Expression of the specific mRNA for the calcitonin receptor as detected by RT-PCR. U2OS cells show a positive band at the expected size (386 bp) ( lane 1 ), whereas another human osteosarcoma

    Article Snippet: The osteosarcoma cell lines U2OS and Saos-2 and the breast cancer cell line T47D (all from the American Type Culture Collection catalog; distributed by LGC Promochem, Teddington, Middlesex, UK) were cultured in McCoy’s medium with 10% fetal calf serum (Gibco, Paisley, UK), 100 U/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO2 /95% air at 37°C and passaged two times a week.

    Techniques: Expressing, Functional Assay, Reverse Transcription Polymerase Chain Reaction

    Dimerization of NTCP wild-type and S267F NTCP. Co-immunoprecipitation of U2OS parental or HA-NTCP-overexpressing U2OS cells transfected with FLAG®-NTCP-S267F. The immunoprecipitation is shown on the left and the lysate controls are shown on the right. Blot is representative of 2 independent experiments. NTCP, sodium taurocholate co-transporting polypeptide.

    Journal: JHEP Reports

    Article Title: Mechanistic insights into the inhibition of NTCP by myrcludex B

    doi: 10.1016/j.jhepr.2019.07.006

    Figure Lengend Snippet: Dimerization of NTCP wild-type and S267F NTCP. Co-immunoprecipitation of U2OS parental or HA-NTCP-overexpressing U2OS cells transfected with FLAG®-NTCP-S267F. The immunoprecipitation is shown on the left and the lysate controls are shown on the right. Blot is representative of 2 independent experiments. NTCP, sodium taurocholate co-transporting polypeptide.

    Article Snippet: Cell culture U2OS cells (from ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich), supplemented with 10% FCS (Gibco), 1% L-glutamine (Lonza), and 1% penicillin/streptomycin (Lonza).

    Techniques: Immunoprecipitation, Transfection

    Time- and dose-dependent Myrcludex B binding to NTCP. (A) Fluorescence intensity of Myrcludex B-FITC on U2OS-HA-hNTCP cells measured by confocal microscopy directly or 24 h after treatment (scale bar 10 μm). (B) Taurocholate uptake assay in U2OS-HA-hNTCP cells 0 h, 1 h, and 24 h after Myrcludex B treatment. (C) Fluorescence intensity of concentration range of Myrcludex B-FITC on U2OS-HA-hNTCP cells measured by Clariostar. (D) Taurocholate uptake assay in U2OS-HA-hNTCP cells pre-incubated with the indicated concentrations of Myrcludex B for 10 min. (B-D) n = 8 from 2 or 3 independent experiments. All data are represented as mean ± SD. FITC, fluorescein isothiocyanate; HA, hemagglutinin; NTCP, sodium taurocholate co-transporting polypeptide.

    Journal: JHEP Reports

    Article Title: Mechanistic insights into the inhibition of NTCP by myrcludex B

    doi: 10.1016/j.jhepr.2019.07.006

    Figure Lengend Snippet: Time- and dose-dependent Myrcludex B binding to NTCP. (A) Fluorescence intensity of Myrcludex B-FITC on U2OS-HA-hNTCP cells measured by confocal microscopy directly or 24 h after treatment (scale bar 10 μm). (B) Taurocholate uptake assay in U2OS-HA-hNTCP cells 0 h, 1 h, and 24 h after Myrcludex B treatment. (C) Fluorescence intensity of concentration range of Myrcludex B-FITC on U2OS-HA-hNTCP cells measured by Clariostar. (D) Taurocholate uptake assay in U2OS-HA-hNTCP cells pre-incubated with the indicated concentrations of Myrcludex B for 10 min. (B-D) n = 8 from 2 or 3 independent experiments. All data are represented as mean ± SD. FITC, fluorescein isothiocyanate; HA, hemagglutinin; NTCP, sodium taurocholate co-transporting polypeptide.

    Article Snippet: Cell culture U2OS cells (from ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich), supplemented with 10% FCS (Gibco), 1% L-glutamine (Lonza), and 1% penicillin/streptomycin (Lonza).

    Techniques: Binding Assay, Fluorescence, Confocal Microscopy, Concentration Assay, Incubation

    Disappearance rate and internalization of Myrcludex B. (A-B) Representative western blots (A) and quantification (B) of NTCP protein expression in a pulse-chase experiment using biotin in parental U2OS (P) and U2OS-HA-hNTCP cells. Western blots are representative of 5 independent experiments. Full uncropped blots are shown in Fig. S1. (C) Fluorescence intensity of Myrcludex B-FITC on U2OS-HA-hNTCP cells measured by Clariostar. (D) NTCP protein expression in U2OS-HA-hNTCP cells treated with vehicle or 200 nM Myrcludex B prior to the pulse chase experiment. Western blots are representative of 2 independent experiments. All data are represented as mean ± SD, * p ≪0.05 compared to the control determined by one-way ANOVA with Bonferroni post hoc analysis. FITC, fluorescein isothiocyanate; HA, hemagglutinin; NTCP, sodium taurocholate co-transporting polypeptide.

    Journal: JHEP Reports

    Article Title: Mechanistic insights into the inhibition of NTCP by myrcludex B

    doi: 10.1016/j.jhepr.2019.07.006

    Figure Lengend Snippet: Disappearance rate and internalization of Myrcludex B. (A-B) Representative western blots (A) and quantification (B) of NTCP protein expression in a pulse-chase experiment using biotin in parental U2OS (P) and U2OS-HA-hNTCP cells. Western blots are representative of 5 independent experiments. Full uncropped blots are shown in Fig. S1. (C) Fluorescence intensity of Myrcludex B-FITC on U2OS-HA-hNTCP cells measured by Clariostar. (D) NTCP protein expression in U2OS-HA-hNTCP cells treated with vehicle or 200 nM Myrcludex B prior to the pulse chase experiment. Western blots are representative of 2 independent experiments. All data are represented as mean ± SD, * p ≪0.05 compared to the control determined by one-way ANOVA with Bonferroni post hoc analysis. FITC, fluorescein isothiocyanate; HA, hemagglutinin; NTCP, sodium taurocholate co-transporting polypeptide.

    Article Snippet: Cell culture U2OS cells (from ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich), supplemented with 10% FCS (Gibco), 1% L-glutamine (Lonza), and 1% penicillin/streptomycin (Lonza).

    Techniques: Western Blot, Expressing, Pulse Chase, Fluorescence

    Pre-bound Myrcludex B can dissociate from NTCP. Competitive binding of unlabeled Myrcludex B to U2OS-HA-hNTCP cells initially labeled with Myrcludex B-FITC. Fluorescence intensity was measured after 1 h competition. n = 8 from 2 independent experiments. All data are represented as mean ± SD, * p ≪0.05 compared to the control determined by one-way ANOVA with Bonferroni post hoc analysis. FITC, fluorescein isothiocyanate; HA, hemagglutinin; NTCP, sodium taurocholate co-transporting polypeptide.

    Journal: JHEP Reports

    Article Title: Mechanistic insights into the inhibition of NTCP by myrcludex B

    doi: 10.1016/j.jhepr.2019.07.006

    Figure Lengend Snippet: Pre-bound Myrcludex B can dissociate from NTCP. Competitive binding of unlabeled Myrcludex B to U2OS-HA-hNTCP cells initially labeled with Myrcludex B-FITC. Fluorescence intensity was measured after 1 h competition. n = 8 from 2 independent experiments. All data are represented as mean ± SD, * p ≪0.05 compared to the control determined by one-way ANOVA with Bonferroni post hoc analysis. FITC, fluorescein isothiocyanate; HA, hemagglutinin; NTCP, sodium taurocholate co-transporting polypeptide.

    Article Snippet: Cell culture U2OS cells (from ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich), supplemented with 10% FCS (Gibco), 1% L-glutamine (Lonza), and 1% penicillin/streptomycin (Lonza).

    Techniques: Binding Assay, Labeling, Fluorescence

    Effects of JW74 treatment on AXIN2 and TNKS protein levels in OS cells. (A) Total cell lysates from KPD, U2OS, or SaOS-2 cells extracted following 72 h treatment with 0.1% DMSO (control) or 10 μ mol/L JW74 were analyzed by Western blotting using antibodies against AXIN2, TNKS1/2, and ACTIN (loading control). (B) U2OS total cell lysates generated following 24, 48, or 72 h treatment with 10 μ mol/L JW74 or 0.1% DMSO (control) were analyzed by Western blotting, showing that AXIN2 protein levels are elevated by 24 h and remain so 48 and 72 h following drug treatment. (C) U2OS cells were treated with 0.1% DMSO (control) or JW74 (0.5–10 μ mol/L) for 48 h, demonstrating dose-response stabilization of AXIN2. OS, osteosarcoma.

    Journal: Cancer Medicine

    Article Title: The tankyrase-specific inhibitor JW74 affects cell cycle progression and induces apoptosis and differentiation in osteosarcoma cell lines

    doi: 10.1002/cam4.170

    Figure Lengend Snippet: Effects of JW74 treatment on AXIN2 and TNKS protein levels in OS cells. (A) Total cell lysates from KPD, U2OS, or SaOS-2 cells extracted following 72 h treatment with 0.1% DMSO (control) or 10 μ mol/L JW74 were analyzed by Western blotting using antibodies against AXIN2, TNKS1/2, and ACTIN (loading control). (B) U2OS total cell lysates generated following 24, 48, or 72 h treatment with 10 μ mol/L JW74 or 0.1% DMSO (control) were analyzed by Western blotting, showing that AXIN2 protein levels are elevated by 24 h and remain so 48 and 72 h following drug treatment. (C) U2OS cells were treated with 0.1% DMSO (control) or JW74 (0.5–10 μ mol/L) for 48 h, demonstrating dose-response stabilization of AXIN2. OS, osteosarcoma.

    Article Snippet: Cell lines, culture conditions, and reagents The cell lines U2OS, SaOS-2 (both from American type culture collection [ATCC]), and KPD were cultured in RPMI-1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (PAA laboratories Gmbh, Pashing, Austria), glutamax, and penicillin/streptomycin (both from Life Technologies).

    Techniques: Western Blot, Generated

    JW74 treatment inhibits osteosarcoma (OS) growth. (A) The proliferative capacity of KPD, U2OS and SaOS-2 was inhibited following treatment with JW74 (1–10 μ mol/L). Cell densities were measured by IncuCyte live cell imaging. DMSO was included as control. (B) The number of Caspase-3-expressing cells per well, following 52 h exposure to drug was determined using the IncuCyte live cell imaging system. Caspase-3 activity was significantly increased in a dose-dependent manner (* P = 0.014; ** P = 0.008; *** P

    Journal: Cancer Medicine

    Article Title: The tankyrase-specific inhibitor JW74 affects cell cycle progression and induces apoptosis and differentiation in osteosarcoma cell lines

    doi: 10.1002/cam4.170

    Figure Lengend Snippet: JW74 treatment inhibits osteosarcoma (OS) growth. (A) The proliferative capacity of KPD, U2OS and SaOS-2 was inhibited following treatment with JW74 (1–10 μ mol/L). Cell densities were measured by IncuCyte live cell imaging. DMSO was included as control. (B) The number of Caspase-3-expressing cells per well, following 52 h exposure to drug was determined using the IncuCyte live cell imaging system. Caspase-3 activity was significantly increased in a dose-dependent manner (* P = 0.014; ** P = 0.008; *** P

    Article Snippet: Cell lines, culture conditions, and reagents The cell lines U2OS, SaOS-2 (both from American type culture collection [ATCC]), and KPD were cultured in RPMI-1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (PAA laboratories Gmbh, Pashing, Austria), glutamax, and penicillin/streptomycin (both from Life Technologies).

    Techniques: Live Cell Imaging, Expressing, Activity Assay

    JW74 treatment reduces nuclear active β -catenin levels and inhibits transcription of downstream targets. (A) Cytoplasmic and nuclear fractions extracted from U2OS cells following 48 h treatment with 0.1% DMSO (control) or 10 μ mol/L JW74 were analyzed by Western blotting using antibodies against active β -catenin, total β -catenin, ACTIN, or LAMINB1 (loading controls). (B) TCF/LEF reporter assays demonstrate that JW74 inhibits β -catenin mediated activity in U2OS cells. Cells transfected with pTA-Luc-STF and Renilla plasmids were treated with 0.1% DMSO (control) or JW74 (0.1–10 μ mol/L) for 48 h. Data are normalized to Renilla. Significantly decreased reporter activity was observed following treatment with 10 μ mol/L JW74 (* P = 0.033) and 5 μ mol/L JW74 (* P = 0.024). (C) AXIN2 mRNA levels were significantly reduced following JW74 treatments of U2OS cells for 48 h (*5 μ mol/L JW74: P = 0.005 and 10 μ mol/L JW74: P = 0.042) and 72 h (**5 μ mol/L and 10 μ mol/L P

    Journal: Cancer Medicine

    Article Title: The tankyrase-specific inhibitor JW74 affects cell cycle progression and induces apoptosis and differentiation in osteosarcoma cell lines

    doi: 10.1002/cam4.170

    Figure Lengend Snippet: JW74 treatment reduces nuclear active β -catenin levels and inhibits transcription of downstream targets. (A) Cytoplasmic and nuclear fractions extracted from U2OS cells following 48 h treatment with 0.1% DMSO (control) or 10 μ mol/L JW74 were analyzed by Western blotting using antibodies against active β -catenin, total β -catenin, ACTIN, or LAMINB1 (loading controls). (B) TCF/LEF reporter assays demonstrate that JW74 inhibits β -catenin mediated activity in U2OS cells. Cells transfected with pTA-Luc-STF and Renilla plasmids were treated with 0.1% DMSO (control) or JW74 (0.1–10 μ mol/L) for 48 h. Data are normalized to Renilla. Significantly decreased reporter activity was observed following treatment with 10 μ mol/L JW74 (* P = 0.033) and 5 μ mol/L JW74 (* P = 0.024). (C) AXIN2 mRNA levels were significantly reduced following JW74 treatments of U2OS cells for 48 h (*5 μ mol/L JW74: P = 0.005 and 10 μ mol/L JW74: P = 0.042) and 72 h (**5 μ mol/L and 10 μ mol/L P

    Article Snippet: Cell lines, culture conditions, and reagents The cell lines U2OS, SaOS-2 (both from American type culture collection [ATCC]), and KPD were cultured in RPMI-1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (PAA laboratories Gmbh, Pashing, Austria), glutamax, and penicillin/streptomycin (both from Life Technologies).

    Techniques: Western Blot, Activity Assay, Transfection

    JW74 treatment leads to induction of let-7 miRNA. qRT-PCR analyses demonstrating significantly increased (indicated by *) expression of let-7 miRNA orthologs in U2OS cells treated 72 h with JW74 (5 or 10 μ mol/L). Data are normalized to RNU44 expression and relative to control-treated cells (DMSO). Error bars represent standard deviation. qRT-PCR, quantitative real-time polymerase chain reaction.

    Journal: Cancer Medicine

    Article Title: The tankyrase-specific inhibitor JW74 affects cell cycle progression and induces apoptosis and differentiation in osteosarcoma cell lines

    doi: 10.1002/cam4.170

    Figure Lengend Snippet: JW74 treatment leads to induction of let-7 miRNA. qRT-PCR analyses demonstrating significantly increased (indicated by *) expression of let-7 miRNA orthologs in U2OS cells treated 72 h with JW74 (5 or 10 μ mol/L). Data are normalized to RNU44 expression and relative to control-treated cells (DMSO). Error bars represent standard deviation. qRT-PCR, quantitative real-time polymerase chain reaction.

    Article Snippet: Cell lines, culture conditions, and reagents The cell lines U2OS, SaOS-2 (both from American type culture collection [ATCC]), and KPD were cultured in RPMI-1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (PAA laboratories Gmbh, Pashing, Austria), glutamax, and penicillin/streptomycin (both from Life Technologies).

    Techniques: Quantitative RT-PCR, Expressing, Standard Deviation, Real-time Polymerase Chain Reaction

    Long-term JW74 treatment induces cellular differentiation. Cells were treated as indicated, with either 0.1% DMSO only, 10 μ mol/L JW74 only, osteogenic differentiation cocktail combined with DMSO, or osteogenic differentiation cocktail combined with JW74 (10 μ mol/L). Quantitative measurements of ALP activity relative to total protein concentration and qualitative alizarin red staining are shown for (A) treated U2OS cells, day 24 and (B) treated SaOS-2 cells, day 12. Statistical significant differences in ALP levels are indicated by (*). Error bars represent standard deviation. ALP, alkaline phosphatase.

    Journal: Cancer Medicine

    Article Title: The tankyrase-specific inhibitor JW74 affects cell cycle progression and induces apoptosis and differentiation in osteosarcoma cell lines

    doi: 10.1002/cam4.170

    Figure Lengend Snippet: Long-term JW74 treatment induces cellular differentiation. Cells were treated as indicated, with either 0.1% DMSO only, 10 μ mol/L JW74 only, osteogenic differentiation cocktail combined with DMSO, or osteogenic differentiation cocktail combined with JW74 (10 μ mol/L). Quantitative measurements of ALP activity relative to total protein concentration and qualitative alizarin red staining are shown for (A) treated U2OS cells, day 24 and (B) treated SaOS-2 cells, day 12. Statistical significant differences in ALP levels are indicated by (*). Error bars represent standard deviation. ALP, alkaline phosphatase.

    Article Snippet: Cell lines, culture conditions, and reagents The cell lines U2OS, SaOS-2 (both from American type culture collection [ATCC]), and KPD were cultured in RPMI-1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (PAA laboratories Gmbh, Pashing, Austria), glutamax, and penicillin/streptomycin (both from Life Technologies).

    Techniques: Cell Differentiation, ALP Assay, Activity Assay, Protein Concentration, Staining, Standard Deviation

    Caspase-2 activation in U2OS cells during mitotic arrest is required for apoptosis. ( a ) Caspase-2 Venus-tagged constructs used for BiFC assay and representative images showing caspase-2 activation at 24 h following PLK1-I treatment of U2OS cells. Scale bar=20μm. Quantitation of BiFC fluorescence is indicated. *** P

    Journal: Oncogene

    Article Title: Caspase-2-mediated cell death is required for deleting aneuploid cells

    doi: 10.1038/onc.2016.423

    Figure Lengend Snippet: Caspase-2 activation in U2OS cells during mitotic arrest is required for apoptosis. ( a ) Caspase-2 Venus-tagged constructs used for BiFC assay and representative images showing caspase-2 activation at 24 h following PLK1-I treatment of U2OS cells. Scale bar=20μm. Quantitation of BiFC fluorescence is indicated. *** P

    Article Snippet: The human osteosarcoma (U2OS) cell line (obtained from ATCC) were maintained at 10% CO2 in DMEM media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (JRH Biosciences), 0.2 mM l-glutamine (Sigma-Aldrich), 15 mM HEPES (Sigma-Aldrich) and 100 μM penicillin/streptomycin (Sigma-Aldrich).

    Techniques: Activation Assay, Construct, Bimolecular Fluorescence Complementation Assay, Quantitation Assay, Fluorescence

    Acute knockdown of CASP2 affects cell death and promotes accumulation of cells with multiple and abnormal nuclei following PLK1-I addition. ( a ) Representative images from live cell imaging of GFP-tagged histone H2B expressing U2OS cells treated with Control siRNA or CASP2 siRNA together with PLK1-I at the indicated time points, displaying dead cells (arrows). Scale bar=50 μm. ( b ) Quantitation of dead cells in U2OS Control siRNA or CASP2 siRNA at indicated time points. Data represented as mean±s.d. from three independent experiments. P values are indicated with ** P

    Journal: Oncogene

    Article Title: Caspase-2-mediated cell death is required for deleting aneuploid cells

    doi: 10.1038/onc.2016.423

    Figure Lengend Snippet: Acute knockdown of CASP2 affects cell death and promotes accumulation of cells with multiple and abnormal nuclei following PLK1-I addition. ( a ) Representative images from live cell imaging of GFP-tagged histone H2B expressing U2OS cells treated with Control siRNA or CASP2 siRNA together with PLK1-I at the indicated time points, displaying dead cells (arrows). Scale bar=50 μm. ( b ) Quantitation of dead cells in U2OS Control siRNA or CASP2 siRNA at indicated time points. Data represented as mean±s.d. from three independent experiments. P values are indicated with ** P

    Article Snippet: The human osteosarcoma (U2OS) cell line (obtained from ATCC) were maintained at 10% CO2 in DMEM media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (JRH Biosciences), 0.2 mM l-glutamine (Sigma-Aldrich), 15 mM HEPES (Sigma-Aldrich) and 100 μM penicillin/streptomycin (Sigma-Aldrich).

    Techniques: Live Cell Imaging, Expressing, Quantitation Assay

    Visualization of endogenous transcription factors and phosphorylated H2AX with VANIMA. (A) The labeled mAbs binding specifically to the transcription factors RPB1, TAF10, and TBP were transduced in U2OS cells, and their localization in the cells was monitored by confocal microscopy 24 h after treatment. A single z plane is shown for each condition. The pictures represent a typical nucleus recorded in each case after fixation of the cells and subsequent counterstaining with DAPI. (B) Same as in A, except that the experiments were performed with the corresponding labeled Fab fragments. (C) Increasing amounts of Alexa Fluor 488–labeled anti-TAF10 mAb (green) were transduced in U2OS cells and fixed 24 h after electroporation (anti-TAF10 Electroporation). To verify binding of the antibody to TAF10, a competition assay was performed afterward by adding a constant amount (2 µg) of the same antibody but Alexa Fluor 568–labeled as IF antibody (red, anti-TAF10 IF; see also Fig. S1 C for quantification). DAPI staining is shown in gray. (D) The labeled Fab raised against γH2AX was transduced as in B, and its localization was recorded after treatment of the electroporated cells with either NCS (for 15 min) or HU (for 48 h). Control, nontreated cells. A typical nucleus is represented in each case. (E) After transduction with Alexa Fluor 488–labeled anti-γH2AX Fab (5 µg) and treatment with HU, cells were treated with or without CSK buffer before fixation. The histogram shows the mean fluorescence intensity of the nucleus of nontreated (−CSK) and CSK-treated (+CSK) cells 24 h (Elec 24h) or 48 h (Elec 48h) after electroporation. The +CSK signal is represented as the percentage of the mean intensity of the −CSK signal. Error bars represent the SD obtained with 10 recorded cells for each condition. Bars, 5 µm.

    Journal: The Journal of Cell Biology

    Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

    doi: 10.1083/jcb.201709153

    Figure Lengend Snippet: Visualization of endogenous transcription factors and phosphorylated H2AX with VANIMA. (A) The labeled mAbs binding specifically to the transcription factors RPB1, TAF10, and TBP were transduced in U2OS cells, and their localization in the cells was monitored by confocal microscopy 24 h after treatment. A single z plane is shown for each condition. The pictures represent a typical nucleus recorded in each case after fixation of the cells and subsequent counterstaining with DAPI. (B) Same as in A, except that the experiments were performed with the corresponding labeled Fab fragments. (C) Increasing amounts of Alexa Fluor 488–labeled anti-TAF10 mAb (green) were transduced in U2OS cells and fixed 24 h after electroporation (anti-TAF10 Electroporation). To verify binding of the antibody to TAF10, a competition assay was performed afterward by adding a constant amount (2 µg) of the same antibody but Alexa Fluor 568–labeled as IF antibody (red, anti-TAF10 IF; see also Fig. S1 C for quantification). DAPI staining is shown in gray. (D) The labeled Fab raised against γH2AX was transduced as in B, and its localization was recorded after treatment of the electroporated cells with either NCS (for 15 min) or HU (for 48 h). Control, nontreated cells. A typical nucleus is represented in each case. (E) After transduction with Alexa Fluor 488–labeled anti-γH2AX Fab (5 µg) and treatment with HU, cells were treated with or without CSK buffer before fixation. The histogram shows the mean fluorescence intensity of the nucleus of nontreated (−CSK) and CSK-treated (+CSK) cells 24 h (Elec 24h) or 48 h (Elec 48h) after electroporation. The +CSK signal is represented as the percentage of the mean intensity of the −CSK signal. Error bars represent the SD obtained with 10 recorded cells for each condition. Bars, 5 µm.

    Article Snippet: Cell culture The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 µg/ml gentamicin.

    Techniques: Labeling, Binding Assay, Confocal Microscopy, Electroporation, Competitive Binding Assay, Staining, Transduction, Fluorescence

    Live imaging of transcription factors by using VANIMA. (A) 24 h after electroporation, U2OS cells transduced with Alexa Fluor 488–labeled anti-RPB1 mAb were subjected to live-cell analysis by confocal microscopy focusing on one z section of individual nuclei. They were imaged over a period of 2.5 h and pictures taken every 10 min (Video 7). Arrows point to two larger Pol II cluster examples that move over time. Bar, 5 µm. (B) Imaging by 3D-SIM microscopy of an individual Pol II cluster observed in U2OS after transduction as in A. The images were taken over a period of 37 s every 4.1 s and show a maximum intensity projection of the 3D video (Video 8). Bar, 1 µm. (C) U2OS cells transduced as in A with the labeled anti-γH2AX Fab were subjected to live-cell analysis by spinning-disk confocal microscopy after the addition of NCS to the culture medium. Pictures were taken every 10 min over a period of 4 h (Video 9) and by focusing on a single z plane. The first time point (0 min) corresponds to the time of the drug addition. Arrows point to γH2AX clusters that appear and disappear over time. Bar, 5 µm. (D) Imaging of an individual γH2AX cluster by 3D-SIM microscopy observed in U2OS cells after transduction as in C. Images were recorded over a period of 45 s every 15 s (Video 10). The first time point (0 s) shown was taken 10 min after NCS treatment. Bar, 0.8 µm.

    Journal: The Journal of Cell Biology

    Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

    doi: 10.1083/jcb.201709153

    Figure Lengend Snippet: Live imaging of transcription factors by using VANIMA. (A) 24 h after electroporation, U2OS cells transduced with Alexa Fluor 488–labeled anti-RPB1 mAb were subjected to live-cell analysis by confocal microscopy focusing on one z section of individual nuclei. They were imaged over a period of 2.5 h and pictures taken every 10 min (Video 7). Arrows point to two larger Pol II cluster examples that move over time. Bar, 5 µm. (B) Imaging by 3D-SIM microscopy of an individual Pol II cluster observed in U2OS after transduction as in A. The images were taken over a period of 37 s every 4.1 s and show a maximum intensity projection of the 3D video (Video 8). Bar, 1 µm. (C) U2OS cells transduced as in A with the labeled anti-γH2AX Fab were subjected to live-cell analysis by spinning-disk confocal microscopy after the addition of NCS to the culture medium. Pictures were taken every 10 min over a period of 4 h (Video 9) and by focusing on a single z plane. The first time point (0 min) corresponds to the time of the drug addition. Arrows point to γH2AX clusters that appear and disappear over time. Bar, 5 µm. (D) Imaging of an individual γH2AX cluster by 3D-SIM microscopy observed in U2OS cells after transduction as in C. Images were recorded over a period of 45 s every 15 s (Video 10). The first time point (0 s) shown was taken 10 min after NCS treatment. Bar, 0.8 µm.

    Article Snippet: Cell culture The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 µg/ml gentamicin.

    Techniques: Imaging, Electroporation, Transduction, Labeling, Confocal Microscopy, Microscopy

    Quantification of transcription factor distribution in single cells by using VANIMA and super-resolution microscopy. (A) U2OS cells were transduced with Alexa Fluor 488–labeled anti-RPB1 mAb and then treated with Flavo (2 µM) for 1 h or not (Untreated). 24 h after treatment the cells were fixed and analyzed by 3D-SIM. The number of individual spots and their volume in individual nuclei were quantified by using Fiji/ImageJ and Matlab software. The graph shows the percentage of spots with a given volume in untreated (red) and treated cells with Flavo (blue) acquired from 10 individual cells for each condition. (B) Same treatment and analysis as in A, but an Alexa Fluor 488–labeled anti-TAF10 antibody was transduced. (C) Spot volumes were extracted from A and B, and the percentage of spots of RPB1 and TAF10 with a volume > 10 −2 μm 3 in the untreated (red) and Flavo (blue) treated cells is shown. The error bars represent the SE from 10 individual cells for each condition. (D) Total number of RPB1 and TAF10 spots in 10 individual nuclei for each condition are represented. (E) Mean cluster size of the RPB1 or TAF10 spots in 10 individual cells for each condition is shown. (F) Total spot volume of RPB1 and TAF10 in 10 individual nuclei for each condition is represented. All black boxes in D–F represent the means and their SEs for each sample. All p-values were calculated by using the two-sample t test.

    Journal: The Journal of Cell Biology

    Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

    doi: 10.1083/jcb.201709153

    Figure Lengend Snippet: Quantification of transcription factor distribution in single cells by using VANIMA and super-resolution microscopy. (A) U2OS cells were transduced with Alexa Fluor 488–labeled anti-RPB1 mAb and then treated with Flavo (2 µM) for 1 h or not (Untreated). 24 h after treatment the cells were fixed and analyzed by 3D-SIM. The number of individual spots and their volume in individual nuclei were quantified by using Fiji/ImageJ and Matlab software. The graph shows the percentage of spots with a given volume in untreated (red) and treated cells with Flavo (blue) acquired from 10 individual cells for each condition. (B) Same treatment and analysis as in A, but an Alexa Fluor 488–labeled anti-TAF10 antibody was transduced. (C) Spot volumes were extracted from A and B, and the percentage of spots of RPB1 and TAF10 with a volume > 10 −2 μm 3 in the untreated (red) and Flavo (blue) treated cells is shown. The error bars represent the SE from 10 individual cells for each condition. (D) Total number of RPB1 and TAF10 spots in 10 individual nuclei for each condition are represented. (E) Mean cluster size of the RPB1 or TAF10 spots in 10 individual cells for each condition is shown. (F) Total spot volume of RPB1 and TAF10 in 10 individual nuclei for each condition is represented. All black boxes in D–F represent the means and their SEs for each sample. All p-values were calculated by using the two-sample t test.

    Article Snippet: Cell culture The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 µg/ml gentamicin.

    Techniques: Microscopy, Transduction, Labeling, Software

    The mAbs do not inhibit premRNA transcription, cell cycle progression, cell proliferation and do not induce apoptosis. (A) U2OS cells electroporated but without antibodies (UT elec), electroporated and treated with α-amanitin (α-ama), electroporated with a control antibody binding to bacterial MBP (anti-MBP), or electroporated with the mAbs recognizing specifically RPB1, TAF10, or TBP (anti-RPB1, anti-TAF10, or anti-TBP). 24 h after electroporation, total RNA was isolated, and the expression of Pol I, Pol II, and Pol III genes was analyzed by RT-qPCR. Pol III transcripts were used for normalization. Newly synthesized RNA of the indicated genes was quantified with validated primer pairs (Table S2). The histograms correspond to the mean values obtained with three independent experiments. (B) The mean values of the three independent experiments shown in A are represented as a heatmap reflecting unchanged relative expression in black, up-regulation in green, and down-regulation in red. (C) U2OS cells were electroporated as in A, and cell cycle progression was monitored by propidium iodide staining and FACS analysis 24 or 48 h after electroporation. The cell cycle phases were normalized to cells electroporated without antibody. (D) U2OS cells were electroporated as in A, and their capacity of proliferation was monitored 24 h after transduction by EdU incorporation and FACS. The electroporated cells without the addition of antibody were used as control. The color code is as in A. (E) The cells were treated as in A, except an apoptosis test was performed 24 h after electroporation. Apoptosis induced by the addition of 10 µM H 2 O 2 was taken as reference (100%). In each panel, the error bars represent the biological SD obtained from three independent replicates. UT, untreated cells.

    Journal: The Journal of Cell Biology

    Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

    doi: 10.1083/jcb.201709153

    Figure Lengend Snippet: The mAbs do not inhibit premRNA transcription, cell cycle progression, cell proliferation and do not induce apoptosis. (A) U2OS cells electroporated but without antibodies (UT elec), electroporated and treated with α-amanitin (α-ama), electroporated with a control antibody binding to bacterial MBP (anti-MBP), or electroporated with the mAbs recognizing specifically RPB1, TAF10, or TBP (anti-RPB1, anti-TAF10, or anti-TBP). 24 h after electroporation, total RNA was isolated, and the expression of Pol I, Pol II, and Pol III genes was analyzed by RT-qPCR. Pol III transcripts were used for normalization. Newly synthesized RNA of the indicated genes was quantified with validated primer pairs (Table S2). The histograms correspond to the mean values obtained with three independent experiments. (B) The mean values of the three independent experiments shown in A are represented as a heatmap reflecting unchanged relative expression in black, up-regulation in green, and down-regulation in red. (C) U2OS cells were electroporated as in A, and cell cycle progression was monitored by propidium iodide staining and FACS analysis 24 or 48 h after electroporation. The cell cycle phases were normalized to cells electroporated without antibody. (D) U2OS cells were electroporated as in A, and their capacity of proliferation was monitored 24 h after transduction by EdU incorporation and FACS. The electroporated cells without the addition of antibody were used as control. The color code is as in A. (E) The cells were treated as in A, except an apoptosis test was performed 24 h after electroporation. Apoptosis induced by the addition of 10 µM H 2 O 2 was taken as reference (100%). In each panel, the error bars represent the biological SD obtained from three independent replicates. UT, untreated cells.

    Article Snippet: Cell culture The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 µg/ml gentamicin.

    Techniques: Binding Assay, Electroporation, Isolation, Expressing, Quantitative RT-PCR, Synthesized, Staining, FACS, Transduction

    Visualization of transcription factors with VANIMA by super-resolution microscopy. (A) The labeled mAbs binding to the transcription factors RPB1, TAF10, and TBP (yellow) were transduced in U2OS cells, and their localization in the cells was monitored 24 h after transduction by 3D-SIM. The pictures show a typical nucleus recorded in each case after fixation and DAPI (gray) treatment (Videos 2–4). The Z maximum intensity projections of five slices show the labeled mAbs with (right half) or without (left half) DAPI counterstaining (gray). The solid white lines depict the nuclear contour. Bottom: Magnification of the white regions of interest, under the corresponding image. (B) The nuclei shown correspond to transduced U2OS cells as in A, except that transductions were performed with the corresponding labeled Fab fragments. Bars, 2 µm.

    Journal: The Journal of Cell Biology

    Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

    doi: 10.1083/jcb.201709153

    Figure Lengend Snippet: Visualization of transcription factors with VANIMA by super-resolution microscopy. (A) The labeled mAbs binding to the transcription factors RPB1, TAF10, and TBP (yellow) were transduced in U2OS cells, and their localization in the cells was monitored 24 h after transduction by 3D-SIM. The pictures show a typical nucleus recorded in each case after fixation and DAPI (gray) treatment (Videos 2–4). The Z maximum intensity projections of five slices show the labeled mAbs with (right half) or without (left half) DAPI counterstaining (gray). The solid white lines depict the nuclear contour. Bottom: Magnification of the white regions of interest, under the corresponding image. (B) The nuclei shown correspond to transduced U2OS cells as in A, except that transductions were performed with the corresponding labeled Fab fragments. Bars, 2 µm.

    Article Snippet: Cell culture The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 µg/ml gentamicin.

    Techniques: Microscopy, Labeling, Binding Assay, Transduction

    Behavior of the anti-RPB1 mAb in U2OS cells. (A) After transduction with Alexa Fluor 488–labeled anti-RPB1 antibodies, cells were imaged after 6 h of incubation and then every hour over a period of 20 h (see Video 1 for all time points). Bar, 15 µm. (B) Increasing amounts of Alexa Fluor 488–labeled anti-RPB1 mAb were transduced in U2OS cells and fixed 24 h after electroporation. A typical nucleus recorded in each case after counterstaining with DAPI is shown. Bar, 5 µm. (C) Binding capacity of anti-RPB1 mAb in U2OS cells. Cells were electroporated with 0 (mock), 0.5, 2, and 4 µg anti-RPB1 mAb and whole-cell extracts prepared 24 h after transduction (INPUT) were mixed with protein G beads. Bound and unbound material was analyzed by Western blotting. The blot shows the fraction of antibody-bound Pol II molecules adsorbed on the beads (beads) or left in the supernatant (SN), and detected with a secondary antibody. (D) After transduction with Alexa Fluor 488–labeled anti-RPB1 mAb (2 µg), cells were treated with or without CSK buffer. The histogram shows the mean fluorescence intensity of the nucleus of nontreated (−CSK) and CSK-treated (+CSK) cells 24 h (Elec 24h) or 48 h (Elec 48h) after electroporation. A classical anti-RPB1 mAb IF experiment was performed as additional control (IF). The +CSK signal is represented as the percentage of the mean intensity of the −CSK signal. Error bars represent the SD obtained with 10 recorded cells for each condition. All images were acquired by confocal microscopy on one single z plane.

    Journal: The Journal of Cell Biology

    Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

    doi: 10.1083/jcb.201709153

    Figure Lengend Snippet: Behavior of the anti-RPB1 mAb in U2OS cells. (A) After transduction with Alexa Fluor 488–labeled anti-RPB1 antibodies, cells were imaged after 6 h of incubation and then every hour over a period of 20 h (see Video 1 for all time points). Bar, 15 µm. (B) Increasing amounts of Alexa Fluor 488–labeled anti-RPB1 mAb were transduced in U2OS cells and fixed 24 h after electroporation. A typical nucleus recorded in each case after counterstaining with DAPI is shown. Bar, 5 µm. (C) Binding capacity of anti-RPB1 mAb in U2OS cells. Cells were electroporated with 0 (mock), 0.5, 2, and 4 µg anti-RPB1 mAb and whole-cell extracts prepared 24 h after transduction (INPUT) were mixed with protein G beads. Bound and unbound material was analyzed by Western blotting. The blot shows the fraction of antibody-bound Pol II molecules adsorbed on the beads (beads) or left in the supernatant (SN), and detected with a secondary antibody. (D) After transduction with Alexa Fluor 488–labeled anti-RPB1 mAb (2 µg), cells were treated with or without CSK buffer. The histogram shows the mean fluorescence intensity of the nucleus of nontreated (−CSK) and CSK-treated (+CSK) cells 24 h (Elec 24h) or 48 h (Elec 48h) after electroporation. A classical anti-RPB1 mAb IF experiment was performed as additional control (IF). The +CSK signal is represented as the percentage of the mean intensity of the −CSK signal. Error bars represent the SD obtained with 10 recorded cells for each condition. All images were acquired by confocal microscopy on one single z plane.

    Article Snippet: Cell culture The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 µg/ml gentamicin.

    Techniques: Transduction, Labeling, Incubation, Electroporation, Binding Assay, Western Blot, Fluorescence, Confocal Microscopy

    Imaging of phosphorylated H2AX with VANIMA by super-resolution microscopy. (A) The labeled anti-γH2AX Fab (yellow) was transduced in U2OS cells, and its localization in the nucleus was recorded by 3D-SIM after treatment with HU for 48 h (+HU) and staining with DAPI (gray). Untreated cells (−HU) were used as the control. The Z maximum intensity projections of 20 slices show the labeled anti-γH2AX Fab with (right half) or without (left half) DAPI counterstaining (gray). The solid white lines depict the nuclear contour. Bottom panels: magnification of the white regions of interest, under the corresponding image (Videos 5 and 6). Bars, 2 µm. (B) The number of spots presented in the nuclei as shown in A after quantification with Fiji/ImageJ software. Error bars represent the SD obtained with five recorded cells for each condition.

    Journal: The Journal of Cell Biology

    Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

    doi: 10.1083/jcb.201709153

    Figure Lengend Snippet: Imaging of phosphorylated H2AX with VANIMA by super-resolution microscopy. (A) The labeled anti-γH2AX Fab (yellow) was transduced in U2OS cells, and its localization in the nucleus was recorded by 3D-SIM after treatment with HU for 48 h (+HU) and staining with DAPI (gray). Untreated cells (−HU) were used as the control. The Z maximum intensity projections of 20 slices show the labeled anti-γH2AX Fab with (right half) or without (left half) DAPI counterstaining (gray). The solid white lines depict the nuclear contour. Bottom panels: magnification of the white regions of interest, under the corresponding image (Videos 5 and 6). Bars, 2 µm. (B) The number of spots presented in the nuclei as shown in A after quantification with Fiji/ImageJ software. Error bars represent the SD obtained with five recorded cells for each condition.

    Article Snippet: Cell culture The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 µg/ml gentamicin.

    Techniques: Imaging, Microscopy, Labeling, Staining, Software

    Inhibition of NHE1 decreased the ROS-induced migration of OS cells. pHe (A), immunoblots of MMP-2 and MMP-9 (B), wound healing ability (C), and invasion (D) were analyzed after treating U2OS cells with 1 μM tBHP alone or in combination with DMTU or cariporide. Data represent mean ± SD of N = 5. *P

    Journal: Journal of Clinical and Translational Research

    Article Title: Osteosarcoma cell proliferation and migration are partly regulated by redox-activated NHE-1

    doi:

    Figure Lengend Snippet: Inhibition of NHE1 decreased the ROS-induced migration of OS cells. pHe (A), immunoblots of MMP-2 and MMP-9 (B), wound healing ability (C), and invasion (D) were analyzed after treating U2OS cells with 1 μM tBHP alone or in combination with DMTU or cariporide. Data represent mean ± SD of N = 5. *P

    Article Snippet: Cell culture and treatment The human OS cell lines U2OS, HOS, HOS-143B, and KHOS as well as the osteoblast cell line hFOB 1.19 were purchased from American Type Culture Collection (Rockville, MD).

    Techniques: Inhibition, Migration, Western Blot

    ROS induces proliferation in U2OS cells and activates NHE1 dose-dependently. Cell viability (A), cell cycle (B), apoptosis (C), and NHE1 protein expression (D) were analyzed after incubation with increasing concentrations of the ROS precursor tBHP. In the flow cytograms of (C), a cell population in the upper right corner (PI- and annexin V-positive) is characteristic of late apoptotic cells, whereas a cell population in the lower right corner (PI-negative and annexin V-positive) typifies early apoptotic cells. The quantification was performed on right upper and lower quadrant cells. Data represent mean ± SD of N = 5. *P

    Journal: Journal of Clinical and Translational Research

    Article Title: Osteosarcoma cell proliferation and migration are partly regulated by redox-activated NHE-1

    doi:

    Figure Lengend Snippet: ROS induces proliferation in U2OS cells and activates NHE1 dose-dependently. Cell viability (A), cell cycle (B), apoptosis (C), and NHE1 protein expression (D) were analyzed after incubation with increasing concentrations of the ROS precursor tBHP. In the flow cytograms of (C), a cell population in the upper right corner (PI- and annexin V-positive) is characteristic of late apoptotic cells, whereas a cell population in the lower right corner (PI-negative and annexin V-positive) typifies early apoptotic cells. The quantification was performed on right upper and lower quadrant cells. Data represent mean ± SD of N = 5. *P

    Article Snippet: Cell culture and treatment The human OS cell lines U2OS, HOS, HOS-143B, and KHOS as well as the osteoblast cell line hFOB 1.19 were purchased from American Type Culture Collection (Rockville, MD).

    Techniques: Expressing, Incubation, Flow Cytometry

    Inhibition of NHE1 decreased ROS-induced OS cell proliferation. Intracellular ROS (A), cell cycle (B), pHi (C), and immunoblots of pERK, Ki67, and NHE1 (D) were assayed after treating U2OS cells with 1 μM tBHP alone or in combination with DMTU or cariporide. Data represent mean ± SD of N = 5. *P

    Journal: Journal of Clinical and Translational Research

    Article Title: Osteosarcoma cell proliferation and migration are partly regulated by redox-activated NHE-1

    doi:

    Figure Lengend Snippet: Inhibition of NHE1 decreased ROS-induced OS cell proliferation. Intracellular ROS (A), cell cycle (B), pHi (C), and immunoblots of pERK, Ki67, and NHE1 (D) were assayed after treating U2OS cells with 1 μM tBHP alone or in combination with DMTU or cariporide. Data represent mean ± SD of N = 5. *P

    Article Snippet: Cell culture and treatment The human OS cell lines U2OS, HOS, HOS-143B, and KHOS as well as the osteoblast cell line hFOB 1.19 were purchased from American Type Culture Collection (Rockville, MD).

    Techniques: Inhibition, Western Blot

    TAT-RasGAP 317−326 , but not mutated or truncated forms, has antimicrobial activities. (A) Contaminated U2OS cell cultures treated or not with 20 μM TAT-RasGAP 317−326 . Please observe the yellow color due to lower pH in presence of the growing contaminant. The contaminant was identified as Staphylococcus capitis . The panel on the right shows the sensitivity of this isolate to the peptide. The bacteria were grown in the presence of the indicated concentrations of TAT-RasGAP 317−326 . The OD 595 nm was measured after 16 h of incubation at 37°C. IC 50 , fifty percent maximal inhibitory concentration; MIC, Minimal inhibitory concentration. (B) E. coli DH5α [optical density (OD) 600 nm of 0.25] were incubated for 7 h at 37°C with the indicated concentrations of WT, mutated (W317A), or truncated TAT-RasGAP 317−326 (RasGAP 317−326 and TAT-RasGAP 317−319 ) peptides. The OD 600 nm was then measured. The results correspond to the mean ± 95% CI of three independent experiments. (C) E. coli DH5α (OD 600 nm of 0.25) were treated with 30 μM of different truncated versions of TAT-RasGAP 317−326 for 7 h at 37°C at which time bacterial density was recorded (OD 600 nm). White box represents a missing residue. Sequences highlighted in green allow at least 80% decrease of OD 600 nm compared to untreated condition. Sequences highlighted in red do not inhibit E. coli growth. The results are derived from three independent experiments. (D) E. coli DH5α (OD 600 nm of 0.25) were treated or not with 20 μM of TAT-RasGAP 317−326 for the indicated periods of time at 37°C. Bacteria were diluted in bacterial culture medium without peptide and colony forming units (CFUs) were determined on agar plates. The results correspond to the mean ± 95% CI of three independent experiments. (E) E. coli DH5α (OD 600 nm of 0.25) were treated or not (NT) with 20 μM of TAT-RasGAP 317−326 (TP) for 6 h at 37°C. The percent of live cells was determined using LIVE/DEAD kit. The results correspond to the mean ± 95% CI of three independent experiments. (F) U2OS cells were incubated 3 days with the supernatant of a Mycoplasma hyorhinis -infected cell culture. Then, 30,000 cells were plated in a 6-well plate and 24 h later treated with 0, 20, and 40 μM of TAT-RasGAP 317−326 for 3 additional days. The medium was then replaced with fresh peptide-containing medium and the cells incubated for 10 more days. The supernatant of the culture was used to detect the presence of mycoplasma DNA by PCR as previously described (Uphoff and Drexler, 2005 ).

    Journal: Frontiers in Microbiology

    Article Title: The Anticancer Peptide TAT-RasGAP317−326 Exerts Broad Antimicrobial Activity

    doi: 10.3389/fmicb.2017.00994

    Figure Lengend Snippet: TAT-RasGAP 317−326 , but not mutated or truncated forms, has antimicrobial activities. (A) Contaminated U2OS cell cultures treated or not with 20 μM TAT-RasGAP 317−326 . Please observe the yellow color due to lower pH in presence of the growing contaminant. The contaminant was identified as Staphylococcus capitis . The panel on the right shows the sensitivity of this isolate to the peptide. The bacteria were grown in the presence of the indicated concentrations of TAT-RasGAP 317−326 . The OD 595 nm was measured after 16 h of incubation at 37°C. IC 50 , fifty percent maximal inhibitory concentration; MIC, Minimal inhibitory concentration. (B) E. coli DH5α [optical density (OD) 600 nm of 0.25] were incubated for 7 h at 37°C with the indicated concentrations of WT, mutated (W317A), or truncated TAT-RasGAP 317−326 (RasGAP 317−326 and TAT-RasGAP 317−319 ) peptides. The OD 600 nm was then measured. The results correspond to the mean ± 95% CI of three independent experiments. (C) E. coli DH5α (OD 600 nm of 0.25) were treated with 30 μM of different truncated versions of TAT-RasGAP 317−326 for 7 h at 37°C at which time bacterial density was recorded (OD 600 nm). White box represents a missing residue. Sequences highlighted in green allow at least 80% decrease of OD 600 nm compared to untreated condition. Sequences highlighted in red do not inhibit E. coli growth. The results are derived from three independent experiments. (D) E. coli DH5α (OD 600 nm of 0.25) were treated or not with 20 μM of TAT-RasGAP 317−326 for the indicated periods of time at 37°C. Bacteria were diluted in bacterial culture medium without peptide and colony forming units (CFUs) were determined on agar plates. The results correspond to the mean ± 95% CI of three independent experiments. (E) E. coli DH5α (OD 600 nm of 0.25) were treated or not (NT) with 20 μM of TAT-RasGAP 317−326 (TP) for 6 h at 37°C. The percent of live cells was determined using LIVE/DEAD kit. The results correspond to the mean ± 95% CI of three independent experiments. (F) U2OS cells were incubated 3 days with the supernatant of a Mycoplasma hyorhinis -infected cell culture. Then, 30,000 cells were plated in a 6-well plate and 24 h later treated with 0, 20, and 40 μM of TAT-RasGAP 317−326 for 3 additional days. The medium was then replaced with fresh peptide-containing medium and the cells incubated for 10 more days. The supernatant of the culture was used to detect the presence of mycoplasma DNA by PCR as previously described (Uphoff and Drexler, 2005 ).

    Article Snippet: Cells and mycoplasma The U2OS human osteosarcoma cell line (ATCC® HTB-96™) was cultured in DMEM (Invitrogen, ref. no. 61965) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, ref. no. 10270-106) in 5% CO2 at 37°C.

    Techniques: Incubation, Concentration Assay, Derivative Assay, Infection, Cell Culture, Polymerase Chain Reaction

    Mapping of HPV11 TSSs and splice junctions in U2OS cells via 5′ RACE. U2OS cells were transfected as follows: 500 ng of the HPV11wt genome (lane 1), 500 ng of the HPV11wt genome together with 2 μg of the linearized pBabe-Neo construct (lanes 2 and 3), 500 ng of the HPV11E8- genome (lane 4), or mock transfection (lane 5). PolyA + mRNA was extracted at 4 and 10 days post-transfection, and 5′ RACE analysis was performed using the HPV11-specific primers pr855c (A) , pr1265c (B) , pr1558c (C) , pr2891c (D) and pr3692c (E) . RT-PCR analysis was performed using the HPV11-specific primers pr1241 and pr3692c (F) . All distinct PCR products (marked with letters from A to Q; the same designations are used in the map represented in Figure 5 ) were cloned and sequenced for the analysis of TSSs and/or splice junctions.

    Journal: Virology Journal

    Article Title: The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome

    doi: 10.1186/s12985-015-0292-6

    Figure Lengend Snippet: Mapping of HPV11 TSSs and splice junctions in U2OS cells via 5′ RACE. U2OS cells were transfected as follows: 500 ng of the HPV11wt genome (lane 1), 500 ng of the HPV11wt genome together with 2 μg of the linearized pBabe-Neo construct (lanes 2 and 3), 500 ng of the HPV11E8- genome (lane 4), or mock transfection (lane 5). PolyA + mRNA was extracted at 4 and 10 days post-transfection, and 5′ RACE analysis was performed using the HPV11-specific primers pr855c (A) , pr1265c (B) , pr1558c (C) , pr2891c (D) and pr3692c (E) . RT-PCR analysis was performed using the HPV11-specific primers pr1241 and pr3692c (F) . All distinct PCR products (marked with letters from A to Q; the same designations are used in the map represented in Figure 5 ) were cloned and sequenced for the analysis of TSSs and/or splice junctions.

    Article Snippet: Cell lines and transfection U2OS cells obtained from the American Type Culture Collection (ATCC) (number HTB-96) were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum (FCS).

    Techniques: Transfection, Construct, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Clone Assay

    Mapping of HPV11 promoter activity from E6/E7-containing E1 expression plasmids. (A) U2OS cells were transfected with 1 μg of different HPV11 expression plasmids (indicated at top of panel A and schematically introduced in panel B). PolyA + mRNA was extracted at 24 h post-transfection, and 5′ RACE analysis was performed with the HPV11-specific primer pr1265c. The products from a-e were sequenced, and their structures are shown in panel B. Mock-transfected U2OS cells were used as a negative control (lane 5). (B) Schematic map of the 5′ RACE products from the E1 expression plasmids, together with HPV11 E1 L+, L-, Int1 and Int2 expression vector maps. All of the E1 protein-coding transcripts (indicated with letters from a-e, shown at left) are shown, indicating their exons (solid boxes), introns (lines) and potential TSSs in E6 and E7 ORFs (nt numbers).

    Journal: Virology Journal

    Article Title: The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome

    doi: 10.1186/s12985-015-0292-6

    Figure Lengend Snippet: Mapping of HPV11 promoter activity from E6/E7-containing E1 expression plasmids. (A) U2OS cells were transfected with 1 μg of different HPV11 expression plasmids (indicated at top of panel A and schematically introduced in panel B). PolyA + mRNA was extracted at 24 h post-transfection, and 5′ RACE analysis was performed with the HPV11-specific primer pr1265c. The products from a-e were sequenced, and their structures are shown in panel B. Mock-transfected U2OS cells were used as a negative control (lane 5). (B) Schematic map of the 5′ RACE products from the E1 expression plasmids, together with HPV11 E1 L+, L-, Int1 and Int2 expression vector maps. All of the E1 protein-coding transcripts (indicated with letters from a-e, shown at left) are shown, indicating their exons (solid boxes), introns (lines) and potential TSSs in E6 and E7 ORFs (nt numbers).

    Article Snippet: Cell lines and transfection U2OS cells obtained from the American Type Culture Collection (ATCC) (number HTB-96) were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum (FCS).

    Techniques: Activity Assay, Expressing, Transfection, Negative Control, Plasmid Preparation

    Mapping of polyadenylation CSs in the HPV11 genome in U2OS cells via 3´ RACE. U2OS cells were transfected with the HPV11wt genome (A and B, lane 1) or together with the linearized pBabe-Neo construct (A and B, lanes 2 and 3). Mock transfection was used as a negative control (lane 4). PolyA + mRNA was extracted at 4 or 10 days post-transfection. All distinct products were purified, cloned, sequenced and analyzed. (A) 3´ RACE analysis of the HPV11 early region CSs using the HPV11-specific primer pr3392. The indicated 3´ RACE products represent the potential CSs at nt 4384 and nt ~3248. (B) 3´ RACE analysis of the HPV11 late region CSs using the HPV11-specific primer pr7188. The indicated 3´ RACE products represent the potential late CSs at nt 7485, at nt ~7770 and at nt ~7587.

    Journal: Virology Journal

    Article Title: The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome

    doi: 10.1186/s12985-015-0292-6

    Figure Lengend Snippet: Mapping of polyadenylation CSs in the HPV11 genome in U2OS cells via 3´ RACE. U2OS cells were transfected with the HPV11wt genome (A and B, lane 1) or together with the linearized pBabe-Neo construct (A and B, lanes 2 and 3). Mock transfection was used as a negative control (lane 4). PolyA + mRNA was extracted at 4 or 10 days post-transfection. All distinct products were purified, cloned, sequenced and analyzed. (A) 3´ RACE analysis of the HPV11 early region CSs using the HPV11-specific primer pr3392. The indicated 3´ RACE products represent the potential CSs at nt 4384 and nt ~3248. (B) 3´ RACE analysis of the HPV11 late region CSs using the HPV11-specific primer pr7188. The indicated 3´ RACE products represent the potential late CSs at nt 7485, at nt ~7770 and at nt ~7587.

    Article Snippet: Cell lines and transfection U2OS cells obtained from the American Type Culture Collection (ATCC) (number HTB-96) were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum (FCS).

    Techniques: Transfection, Construct, Negative Control, Purification, Clone Assay

    Transient, stable and amplificational replication of the HPV11wt and HPV11E8- genomes in U2OS cells. The mock-transfected cells were used as a negative control (A, lane 7, B, lane 23 and C, lane 10). The linearized HPV11 genome of 100 copies (A, lane 8, B, lane 24 and C, lane 11) and DpnI fragments (C, lane 12) was used as size markers, also indicated with arrows. (A) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-3) or E8- (lanes 4-6) genome. Extrachromosomal DNA was extracted via Hirt lysis at 3, 4 and 5 days post-transfection, digested with HindIII and with DpnI. The replication signal was detected via the Southern blot method with a radiolabelled HPV11 genome probe. (B) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-11) or HPV11E8- (lanes 12-22) genome, together with 2 μg of the linearized pBabe-Neo construct. The transfected cells were selected with the antibiotic G418, and at 10 days post-transfection, the cells were split and cultivated under either subconfluent (lanes 2-6 and 13-17) or confluent conditions (lanes 7-11 and 18-22). Total DNA was extracted at the time points indicated at top of the figure, and 3 μg of each sample was analyzed as indicated in A . (C) Effect of E8˄E1 and E8˄E2 proteins on viral genome replication. U2OS cells were transfected with 500 ng of the HPV11E8- genome with increasing amounts of either the E8˄E1 or E8˄E2 expression plasmid. Total DNA was extracted at 4 days post-transfection, and 3 μg of each sample was analyzed as indicated in A . (D) The quantitation of HPV11wt and E8- genome DNA replication signals at different time points at subconfluent and confluent culture conditions. The signals were normalized to 10 th day time point. Shown is one of two independent experiments.

    Journal: Virology Journal

    Article Title: The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome

    doi: 10.1186/s12985-015-0292-6

    Figure Lengend Snippet: Transient, stable and amplificational replication of the HPV11wt and HPV11E8- genomes in U2OS cells. The mock-transfected cells were used as a negative control (A, lane 7, B, lane 23 and C, lane 10). The linearized HPV11 genome of 100 copies (A, lane 8, B, lane 24 and C, lane 11) and DpnI fragments (C, lane 12) was used as size markers, also indicated with arrows. (A) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-3) or E8- (lanes 4-6) genome. Extrachromosomal DNA was extracted via Hirt lysis at 3, 4 and 5 days post-transfection, digested with HindIII and with DpnI. The replication signal was detected via the Southern blot method with a radiolabelled HPV11 genome probe. (B) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-11) or HPV11E8- (lanes 12-22) genome, together with 2 μg of the linearized pBabe-Neo construct. The transfected cells were selected with the antibiotic G418, and at 10 days post-transfection, the cells were split and cultivated under either subconfluent (lanes 2-6 and 13-17) or confluent conditions (lanes 7-11 and 18-22). Total DNA was extracted at the time points indicated at top of the figure, and 3 μg of each sample was analyzed as indicated in A . (C) Effect of E8˄E1 and E8˄E2 proteins on viral genome replication. U2OS cells were transfected with 500 ng of the HPV11E8- genome with increasing amounts of either the E8˄E1 or E8˄E2 expression plasmid. Total DNA was extracted at 4 days post-transfection, and 3 μg of each sample was analyzed as indicated in A . (D) The quantitation of HPV11wt and E8- genome DNA replication signals at different time points at subconfluent and confluent culture conditions. The signals were normalized to 10 th day time point. Shown is one of two independent experiments.

    Article Snippet: Cell lines and transfection U2OS cells obtained from the American Type Culture Collection (ATCC) (number HTB-96) were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum (FCS).

    Techniques: Transfection, Negative Control, Lysis, Southern Blot, Construct, Expressing, Plasmid Preparation, Quantitation Assay

    HPV11 E1 expression from E1 plasmids and transient replication of the URR-plasmid in the presence of the E1 and E2 proteins. (A, B) Detection of the HA-epitope tagged HPV11 E1 protein from transiently transfected U2OS cells. The cells were transfected with 1-5000 ng of different HPV11 E1 expression plasmids (indicated at the top of the figure). Western blot analysis was performed at the 24 h time point to detect the HPV11 E1 protein (A) and the cellular marker α-tubulin (B) . (C) The HPV11 E1 and E2 proteins initiated DNA replication from the episomal HPV11 URR plasmid in transiently transfected U2OS cells. U2OS cells were co-transfected with 100 ng of the HPV11 URR plasmid, 100 ng of E2 and 1-5000 ng of different E1 expression plasmids. Extrachromosomal DNA was extracted at 24 and 48 h post-transfection via Hirt lysis, and ½ of each sample was analyzed as indicated in Figure 1 A. The replication signal was detected with a radiolabelled HPV11 URR specific probe. Mock-transfected U2OS cells were used as a negative control (lane 27), and 200 pg of the linearized HPV11 URR plasmid (lane 28) was used as a size marker. The linear HPV11 URR and DpnI fragments are indicated with arrows.

    Journal: Virology Journal

    Article Title: The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome

    doi: 10.1186/s12985-015-0292-6

    Figure Lengend Snippet: HPV11 E1 expression from E1 plasmids and transient replication of the URR-plasmid in the presence of the E1 and E2 proteins. (A, B) Detection of the HA-epitope tagged HPV11 E1 protein from transiently transfected U2OS cells. The cells were transfected with 1-5000 ng of different HPV11 E1 expression plasmids (indicated at the top of the figure). Western blot analysis was performed at the 24 h time point to detect the HPV11 E1 protein (A) and the cellular marker α-tubulin (B) . (C) The HPV11 E1 and E2 proteins initiated DNA replication from the episomal HPV11 URR plasmid in transiently transfected U2OS cells. U2OS cells were co-transfected with 100 ng of the HPV11 URR plasmid, 100 ng of E2 and 1-5000 ng of different E1 expression plasmids. Extrachromosomal DNA was extracted at 24 and 48 h post-transfection via Hirt lysis, and ½ of each sample was analyzed as indicated in Figure 1 A. The replication signal was detected with a radiolabelled HPV11 URR specific probe. Mock-transfected U2OS cells were used as a negative control (lane 27), and 200 pg of the linearized HPV11 URR plasmid (lane 28) was used as a size marker. The linear HPV11 URR and DpnI fragments are indicated with arrows.

    Article Snippet: Cell lines and transfection U2OS cells obtained from the American Type Culture Collection (ATCC) (number HTB-96) were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum (FCS).

    Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Marker, Lysis, Negative Control

    Two direct circadian modulators, KL001 (A) and PF-670462 (B), and three indirect circadian modulators, Chir99021 (C), etoposide (D), and SP600125 (E), influence cell motility to various extents. KL001 at concentrations 5, 7, and 12 µM promotes cell motility at T = 6, 12, and 18 hours (A). PF-670462 at 1.5 µM concentration reduces cell motility (B). SP600125 and Chir99021 treatments significantly inhibit cell migration (C, E), while etoposide shows no effect (D) in U2OS cells. Data are representative of three biological replicates; error bars represent standard deviation. Statistical significance was evaluated via Welch’s T test followed by Bonferroni Correction, * P

    Journal: Integrative Cancer Therapies

    Article Title: Oncogenic and Circadian Effects of Small Molecules Directly and Indirectly Targeting the Core Circadian Clock

    doi: 10.1177/1534735420924094

    Figure Lengend Snippet: Two direct circadian modulators, KL001 (A) and PF-670462 (B), and three indirect circadian modulators, Chir99021 (C), etoposide (D), and SP600125 (E), influence cell motility to various extents. KL001 at concentrations 5, 7, and 12 µM promotes cell motility at T = 6, 12, and 18 hours (A). PF-670462 at 1.5 µM concentration reduces cell motility (B). SP600125 and Chir99021 treatments significantly inhibit cell migration (C, E), while etoposide shows no effect (D) in U2OS cells. Data are representative of three biological replicates; error bars represent standard deviation. Statistical significance was evaluated via Welch’s T test followed by Bonferroni Correction, * P

    Article Snippet: DMSO solutions of molecules were added to 1 mL of 4 × 104 cells/mL suspended U2OS cells in warm culture media, followed by addition of 0.6% agarose solution in a 1:1 ratio.

    Techniques: Concentration Assay, Migration, Standard Deviation

    Three indirect circadian modulators, SP600125 (A, B), Chir99021 (C, D), and etoposide (E, F), increase or decrease the period of Bmal1 in U2OS cells. Representative traces are shown; replicate traces (raw and detrended) for each condition are shown in Supplementary Figures S6, S9, and S10 . The lines (B, D, F) represent mean of the data ±1 standard error. Statistical significance was evaluated via Welch’s T test followed by Bonferroni Correction, * P

    Journal: Integrative Cancer Therapies

    Article Title: Oncogenic and Circadian Effects of Small Molecules Directly and Indirectly Targeting the Core Circadian Clock

    doi: 10.1177/1534735420924094

    Figure Lengend Snippet: Three indirect circadian modulators, SP600125 (A, B), Chir99021 (C, D), and etoposide (E, F), increase or decrease the period of Bmal1 in U2OS cells. Representative traces are shown; replicate traces (raw and detrended) for each condition are shown in Supplementary Figures S6, S9, and S10 . The lines (B, D, F) represent mean of the data ±1 standard error. Statistical significance was evaluated via Welch’s T test followed by Bonferroni Correction, * P

    Article Snippet: DMSO solutions of molecules were added to 1 mL of 4 × 104 cells/mL suspended U2OS cells in warm culture media, followed by addition of 0.6% agarose solution in a 1:1 ratio.

    Techniques:

    Schematic of the mechanism underlying the miR-192-5p/USP1 axis in 143B and U2OS cells. miR, microRNA; USP1, ubiquitin-specific protease 1.

    Journal: Oncology Letters

    Article Title: MicroRNA-192-5p suppresses the initiation and progression of osteosarcoma by targeting USP1

    doi: 10.3892/ol.2018.8180

    Figure Lengend Snippet: Schematic of the mechanism underlying the miR-192-5p/USP1 axis in 143B and U2OS cells. miR, microRNA; USP1, ubiquitin-specific protease 1.

    Article Snippet: For cell cycle assay, transfected cells were inoculated in 6-well plates for 24 h. Then 143B and U2OS cells were fixed with 75% cold ethanol at 4°C for 24 h. Subsequently, both cell lines were stained with a propidium iodide (PI; BD Biosciences, San Jose, CA, USA) for 30 min in the dark.

    Techniques:

    miR-192-5p regulated osteosarcoma cell proliferation, apoptosis, migration, invasion and chemo-sensitivity through USP1. (A) The USP1 mRNA expression was detected by reverse transcription-quantitative polymerase chain reaction. (B) The USP1 protein expression was detected by western blot analysis. (C-I) Ectopic expression of USP1 statistically enhanced cell (C and D) proliferation, decreased cell (E and F) chemo-sensitivity, promoted cell (G) migration and (H) invasion (magnification, ×100), and reduced cell (I) apoptosis in 143B and U2OS cells, which could be partially abolished by miR-192-5p following co-transfection with miR-192-5p mimics and USP1. Data are presented as the mean ± standard deviation of 3 independent assays. *P

    Journal: Oncology Letters

    Article Title: MicroRNA-192-5p suppresses the initiation and progression of osteosarcoma by targeting USP1

    doi: 10.3892/ol.2018.8180

    Figure Lengend Snippet: miR-192-5p regulated osteosarcoma cell proliferation, apoptosis, migration, invasion and chemo-sensitivity through USP1. (A) The USP1 mRNA expression was detected by reverse transcription-quantitative polymerase chain reaction. (B) The USP1 protein expression was detected by western blot analysis. (C-I) Ectopic expression of USP1 statistically enhanced cell (C and D) proliferation, decreased cell (E and F) chemo-sensitivity, promoted cell (G) migration and (H) invasion (magnification, ×100), and reduced cell (I) apoptosis in 143B and U2OS cells, which could be partially abolished by miR-192-5p following co-transfection with miR-192-5p mimics and USP1. Data are presented as the mean ± standard deviation of 3 independent assays. *P

    Article Snippet: For cell cycle assay, transfected cells were inoculated in 6-well plates for 24 h. Then 143B and U2OS cells were fixed with 75% cold ethanol at 4°C for 24 h. Subsequently, both cell lines were stained with a propidium iodide (PI; BD Biosciences, San Jose, CA, USA) for 30 min in the dark.

    Techniques: Migration, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cotransfection, Standard Deviation

    Upregulation of miR-192-5p inhibited osteosarcoma cell proliferation and induced cell cycle at the G0/G1 stage and cell apoptosis. (A) Reverse transcription-quantitative polymerase chain reaction was performed to measure the expression of miR-192-5p in 143B and U2OS cells following transfection with miR-192-5p-mimics. Overexpression of miR-192-5p significantly restrained the proliferation of (B) 143B and (C) U2OS cells. (D) The cell cycle assay demonstrated that miR-192-5p mimics could prevent the progression of the cell cycle from the G1 to S phase in 143B and U2OS cells. (E) Elevated expression of miR-192-5p markedly induced 143B and U2OS cells apoptosis. Data are presented as the mean ± standard deviation of 3 independent assays. *P

    Journal: Oncology Letters

    Article Title: MicroRNA-192-5p suppresses the initiation and progression of osteosarcoma by targeting USP1

    doi: 10.3892/ol.2018.8180

    Figure Lengend Snippet: Upregulation of miR-192-5p inhibited osteosarcoma cell proliferation and induced cell cycle at the G0/G1 stage and cell apoptosis. (A) Reverse transcription-quantitative polymerase chain reaction was performed to measure the expression of miR-192-5p in 143B and U2OS cells following transfection with miR-192-5p-mimics. Overexpression of miR-192-5p significantly restrained the proliferation of (B) 143B and (C) U2OS cells. (D) The cell cycle assay demonstrated that miR-192-5p mimics could prevent the progression of the cell cycle from the G1 to S phase in 143B and U2OS cells. (E) Elevated expression of miR-192-5p markedly induced 143B and U2OS cells apoptosis. Data are presented as the mean ± standard deviation of 3 independent assays. *P

    Article Snippet: For cell cycle assay, transfected cells were inoculated in 6-well plates for 24 h. Then 143B and U2OS cells were fixed with 75% cold ethanol at 4°C for 24 h. Subsequently, both cell lines were stained with a propidium iodide (PI; BD Biosciences, San Jose, CA, USA) for 30 min in the dark.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Over Expression, Cell Cycle Assay, Standard Deviation

    Reduced expression of miR-192-5p and elevated expression of USP1 in osteosarcoma tissues and cell lines. (A) miR-192-5p expression was statistically lower in osteosarcoma tissues than in adjacent non-tumor tissues. (B) USP1 expression was significantly higher in osteosarcoma tissues when compared with adjacent non-tumor tissues. (C) There was a negative correlation between miR-192-5p and USP1 in osteosarcoma tissues. (D) The expression of miR-192-5p was downregulated in the osteosarcoma cell lines (143B and U2OS) when compared with the normal human osteoblast cell line (hFOB). (E) The expression of USP1 was increased in the 143B and U2OS cell lines compared with that of osteosarcoma. (F) Kaplan-Meier survival analysis suggested that osteosarcoma patients with low expression of miR-192-5p presented a shorter overall survival. The results are presented as the mean ± standard deviation of 3 independent assays. ***P

    Journal: Oncology Letters

    Article Title: MicroRNA-192-5p suppresses the initiation and progression of osteosarcoma by targeting USP1

    doi: 10.3892/ol.2018.8180

    Figure Lengend Snippet: Reduced expression of miR-192-5p and elevated expression of USP1 in osteosarcoma tissues and cell lines. (A) miR-192-5p expression was statistically lower in osteosarcoma tissues than in adjacent non-tumor tissues. (B) USP1 expression was significantly higher in osteosarcoma tissues when compared with adjacent non-tumor tissues. (C) There was a negative correlation between miR-192-5p and USP1 in osteosarcoma tissues. (D) The expression of miR-192-5p was downregulated in the osteosarcoma cell lines (143B and U2OS) when compared with the normal human osteoblast cell line (hFOB). (E) The expression of USP1 was increased in the 143B and U2OS cell lines compared with that of osteosarcoma. (F) Kaplan-Meier survival analysis suggested that osteosarcoma patients with low expression of miR-192-5p presented a shorter overall survival. The results are presented as the mean ± standard deviation of 3 independent assays. ***P

    Article Snippet: For cell cycle assay, transfected cells were inoculated in 6-well plates for 24 h. Then 143B and U2OS cells were fixed with 75% cold ethanol at 4°C for 24 h. Subsequently, both cell lines were stained with a propidium iodide (PI; BD Biosciences, San Jose, CA, USA) for 30 min in the dark.

    Techniques: Expressing, Standard Deviation

    Overexpression of miR-192-5p suppressed osteosarcoma cell migration and invasion, and enhanced the sensitivity of osteosarcoma cells to cisplatin. (A) Wound-healing assay revealed that the migratory ability of 143B and U2OS cells was suppressed by miR-192-5p mimics (magnification, ×100). (B) Transwell assay was conducted to evaluate the invasion of 143B and U2OS cells following treatment with mR-192-5p, which suggested that the capacity of invasion was significantly decreased in the treatment group (magnification, ×100). (C) The response of 143B cells to cisplatin enhanced following transfection with the miR-192 mimic compared to the negative control. (D) The response of U2OS cells to cisplatin enhanced following transfection with the miR-192 mimic compared with the negative control. Data are presented as the mean ± standard deviation of 3 independent assays. *P

    Journal: Oncology Letters

    Article Title: MicroRNA-192-5p suppresses the initiation and progression of osteosarcoma by targeting USP1

    doi: 10.3892/ol.2018.8180

    Figure Lengend Snippet: Overexpression of miR-192-5p suppressed osteosarcoma cell migration and invasion, and enhanced the sensitivity of osteosarcoma cells to cisplatin. (A) Wound-healing assay revealed that the migratory ability of 143B and U2OS cells was suppressed by miR-192-5p mimics (magnification, ×100). (B) Transwell assay was conducted to evaluate the invasion of 143B and U2OS cells following treatment with mR-192-5p, which suggested that the capacity of invasion was significantly decreased in the treatment group (magnification, ×100). (C) The response of 143B cells to cisplatin enhanced following transfection with the miR-192 mimic compared to the negative control. (D) The response of U2OS cells to cisplatin enhanced following transfection with the miR-192 mimic compared with the negative control. Data are presented as the mean ± standard deviation of 3 independent assays. *P

    Article Snippet: For cell cycle assay, transfected cells were inoculated in 6-well plates for 24 h. Then 143B and U2OS cells were fixed with 75% cold ethanol at 4°C for 24 h. Subsequently, both cell lines were stained with a propidium iodide (PI; BD Biosciences, San Jose, CA, USA) for 30 min in the dark.

    Techniques: Over Expression, Migration, Wound Healing Assay, Transwell Assay, Transfection, Negative Control, Standard Deviation

    USP1 was a direct target of miR-192-5p in osteosarcoma cells. (A) Bioinformatics study suggested that there was a putative binding site between miR-192-5p and USP1. Dual-luciferase reporter system assay indicated that miR-192-5p mimics significantly restrained wild-type 3′UTR-USP1 reporter activity, while there was no repression effect on the mutant 3′UTR-USP1 reporter activity in (B) 143B and (C) U2OS cells. (D) Ectopic expression of miR-192-5p decreased the USP1 mRNA expression in 143B and U2OS cells. (E) Overexpression of miR-192-5p also reduced the expression of USP1 at the protein level. Data are presented as the mean ± standard deviation of 3 independent assays. ***P

    Journal: Oncology Letters

    Article Title: MicroRNA-192-5p suppresses the initiation and progression of osteosarcoma by targeting USP1

    doi: 10.3892/ol.2018.8180

    Figure Lengend Snippet: USP1 was a direct target of miR-192-5p in osteosarcoma cells. (A) Bioinformatics study suggested that there was a putative binding site between miR-192-5p and USP1. Dual-luciferase reporter system assay indicated that miR-192-5p mimics significantly restrained wild-type 3′UTR-USP1 reporter activity, while there was no repression effect on the mutant 3′UTR-USP1 reporter activity in (B) 143B and (C) U2OS cells. (D) Ectopic expression of miR-192-5p decreased the USP1 mRNA expression in 143B and U2OS cells. (E) Overexpression of miR-192-5p also reduced the expression of USP1 at the protein level. Data are presented as the mean ± standard deviation of 3 independent assays. ***P

    Article Snippet: For cell cycle assay, transfected cells were inoculated in 6-well plates for 24 h. Then 143B and U2OS cells were fixed with 75% cold ethanol at 4°C for 24 h. Subsequently, both cell lines were stained with a propidium iodide (PI; BD Biosciences, San Jose, CA, USA) for 30 min in the dark.

    Techniques: Binding Assay, Luciferase, Activity Assay, Mutagenesis, Expressing, Over Expression, Standard Deviation

    Mitotic spindle is chiral. a STED image (single z -plane) of metaphase spindle in a live HeLa cell expressing EGFP-CENP-A and EGFP-centrin1 (both shown in magenta) (left and middle; middle panel shows traces of microtubule bundles superimposed on the image), and in a live U2OS cell expressing CENP-A-GFP (magenta) (right). Microtubules are labeled with SiR-tubulin (green). b Imaging scheme of a vertically oriented spindle. c Imaging plane of a vertical spindle in a fixed HeLa cell expressing PRC1-GFP and mRFP-CENP-B (only PRC1-GFP is shown) (left); orthogonal plane of the same spindle (middle); arrows connecting starting and ending points of PRC1-GFP bundles traced upwards (right). Longer arrows roughly correspond to larger twist around the spindle axis (circle), colors show z -distance between starting and ending points, see color bar in g . d Schematic representation of the microtubule bundle helicity measurement. e Imaging scheme of a horizontally oriented spindle. f Horizontal spindle in a fixed HeLa cell expressing PRC1-GFP and mRFP-CENP-B, legend as in c . g Horizontal spindle in a fixed unlabeled HeLa cell immunostained for PRC1, with DNA stained by DAPI, legend as in c . Images in c left, and f , g middle are single planes; images in c middle, and f , g left are maximum intensity projections of five central planes. h Spindle helicity averaged over bundles for different conditions (vertical and horizontal spindles, fixed and live cells) and cell lines as indicated. Cell lines used were: HeLa cells expressing PRC1-GFP (1st, 2nd, 4th, and 5th bars), unlabeled HeLa cells immunostained for PRC1 (3rd bar), unlabeled U2OS cells immunostained for PRC1 (6th bar), U2OS cells expressing CENP-A-GFP, mCherry-α-tubulin, and photoactivatable (PA)-GFP-tubulin (7th bar). Data are representative of 4 independent experiments for unlabeled HeLa and U2OS cells immunostained for PRC1 and 3 independent experiments for all other conditions. Numbers represent the number of cells (top) and bundles (bottom). Data for individual cells are shown in Supplementary Fig. 1e . i Paper model of the spindle showing left-handed helicity of microtubule bundles and chirality of the whole spindle. Scale bars, 1 μm; error bars, s.e.m

    Journal: Nature Communications

    Article Title: The mitotic spindle is chiral due to torques within microtubule bundles

    doi: 10.1038/s41467-018-06005-7

    Figure Lengend Snippet: Mitotic spindle is chiral. a STED image (single z -plane) of metaphase spindle in a live HeLa cell expressing EGFP-CENP-A and EGFP-centrin1 (both shown in magenta) (left and middle; middle panel shows traces of microtubule bundles superimposed on the image), and in a live U2OS cell expressing CENP-A-GFP (magenta) (right). Microtubules are labeled with SiR-tubulin (green). b Imaging scheme of a vertically oriented spindle. c Imaging plane of a vertical spindle in a fixed HeLa cell expressing PRC1-GFP and mRFP-CENP-B (only PRC1-GFP is shown) (left); orthogonal plane of the same spindle (middle); arrows connecting starting and ending points of PRC1-GFP bundles traced upwards (right). Longer arrows roughly correspond to larger twist around the spindle axis (circle), colors show z -distance between starting and ending points, see color bar in g . d Schematic representation of the microtubule bundle helicity measurement. e Imaging scheme of a horizontally oriented spindle. f Horizontal spindle in a fixed HeLa cell expressing PRC1-GFP and mRFP-CENP-B, legend as in c . g Horizontal spindle in a fixed unlabeled HeLa cell immunostained for PRC1, with DNA stained by DAPI, legend as in c . Images in c left, and f , g middle are single planes; images in c middle, and f , g left are maximum intensity projections of five central planes. h Spindle helicity averaged over bundles for different conditions (vertical and horizontal spindles, fixed and live cells) and cell lines as indicated. Cell lines used were: HeLa cells expressing PRC1-GFP (1st, 2nd, 4th, and 5th bars), unlabeled HeLa cells immunostained for PRC1 (3rd bar), unlabeled U2OS cells immunostained for PRC1 (6th bar), U2OS cells expressing CENP-A-GFP, mCherry-α-tubulin, and photoactivatable (PA)-GFP-tubulin (7th bar). Data are representative of 4 independent experiments for unlabeled HeLa and U2OS cells immunostained for PRC1 and 3 independent experiments for all other conditions. Numbers represent the number of cells (top) and bundles (bottom). Data for individual cells are shown in Supplementary Fig. 1e . i Paper model of the spindle showing left-handed helicity of microtubule bundles and chirality of the whole spindle. Scale bars, 1 μm; error bars, s.e.m

    Article Snippet: To prepare samples for microscopy, HeLa and U2OS cells were seeded and cultured in 1.5 ml DMEM medium with supplements at 37 °C and 5% CO2 on uncoated 35-mm glass coverslip dishes, No. 1.5 coverglass (MatTek Corporation, Ashland, MA, USA).

    Techniques: Expressing, Labeling, Imaging, Staining

    Kif11/Eg5 inactivation by STLC reduces spindle chirality, whereas latrunculin A treatment does not. a Horizontal spindle in a live HeLa cell expressing PRC1-GFP with SiR-DNA-labeled chromosomes, treated with STLC (left); arrows connecting starting and ending points of bundles traced upwards, from the same cell (right). Circle denotes spindle axis, and colors show z -distance between starting and ending points, see color bar. b – e Helicity of spindles before and after STLC or DMSO (mock) treatment, as indicated. c Helicity before treatment was different from zero ( p = 10 −44 ), but not at 5 and 10 min ( p = 0.21 and 0.28). d All helicities were different from zero ( p = 10 −44 , 7 × 10 −9 , and 4 × 10 −9 at 0, 5, and 10 min). f Spindle of a live HeLa cell treated with latrunculin A, legend as in a . g Helicity before and after treatment with latrunculin A. In all panels live HeLa cells expressing PRC1-GFP were used, except in e where live U2OS cells expressing CENP-A-GFP and mCherry-α-tubulin were used. In b – e and g , numbers represent the number of cells (top) and bundles (bottom), from 5 independent experiments in b - e and from 4 independent experiments in g ; *** p

    Journal: Nature Communications

    Article Title: The mitotic spindle is chiral due to torques within microtubule bundles

    doi: 10.1038/s41467-018-06005-7

    Figure Lengend Snippet: Kif11/Eg5 inactivation by STLC reduces spindle chirality, whereas latrunculin A treatment does not. a Horizontal spindle in a live HeLa cell expressing PRC1-GFP with SiR-DNA-labeled chromosomes, treated with STLC (left); arrows connecting starting and ending points of bundles traced upwards, from the same cell (right). Circle denotes spindle axis, and colors show z -distance between starting and ending points, see color bar. b – e Helicity of spindles before and after STLC or DMSO (mock) treatment, as indicated. c Helicity before treatment was different from zero ( p = 10 −44 ), but not at 5 and 10 min ( p = 0.21 and 0.28). d All helicities were different from zero ( p = 10 −44 , 7 × 10 −9 , and 4 × 10 −9 at 0, 5, and 10 min). f Spindle of a live HeLa cell treated with latrunculin A, legend as in a . g Helicity before and after treatment with latrunculin A. In all panels live HeLa cells expressing PRC1-GFP were used, except in e where live U2OS cells expressing CENP-A-GFP and mCherry-α-tubulin were used. In b – e and g , numbers represent the number of cells (top) and bundles (bottom), from 5 independent experiments in b - e and from 4 independent experiments in g ; *** p

    Article Snippet: To prepare samples for microscopy, HeLa and U2OS cells were seeded and cultured in 1.5 ml DMEM medium with supplements at 37 °C and 5% CO2 on uncoated 35-mm glass coverslip dishes, No. 1.5 coverglass (MatTek Corporation, Ashland, MA, USA).

    Techniques: Expressing, Labeling

    Annexin V staining and TUNEL analysis in osteosarcoma cells treated with MTZ. (A) Annexin V staining results of U2OS and MG63 cells treated with MTZ (0, 0.5 and 1 µM) for 2 h. The representative images of the three assays are shown. The percentage of living, necrotic, early and late apoptotic cells are presented in each box of the graph. Graphs with quantitative data were based on the relative ratio of early, late, necrotic and live cells. The error bar indicates standard error. *P

    Journal: Oncology Letters

    Article Title: Mitoxantrone induces apoptosis in osteosarcoma cells through regulation of the Akt/FOXO3 pathway

    doi: 10.3892/ol.2018.8547

    Figure Lengend Snippet: Annexin V staining and TUNEL analysis in osteosarcoma cells treated with MTZ. (A) Annexin V staining results of U2OS and MG63 cells treated with MTZ (0, 0.5 and 1 µM) for 2 h. The representative images of the three assays are shown. The percentage of living, necrotic, early and late apoptotic cells are presented in each box of the graph. Graphs with quantitative data were based on the relative ratio of early, late, necrotic and live cells. The error bar indicates standard error. *P

    Article Snippet: U2OS cells [2×104 /well in 100 µl DMEM (Gibco; Thermo Fisher Scientific, Inc.)] were seeded into 96-well plates and incubated at 37°C for 18 h in a CO2 incubator.

    Techniques: Staining, TUNEL Assay

    FOXO3 dependent-apoptosis in U2OS cells treated with MTZ. Western blotting results of U2OS cells transfected with control or FOXO3 siRNA and treated with MTZ (0 and 0.5 µM) for 48 h, followed by blotting with specific antibodies as indicated. Graphs with quantitative data were based on the relative ratio of the indicated protein to β-actin. MTZ, mitoxantrone; EL, extra-large; FOXO3, forkhead-box O3; PARP1, poly(ADP-ribose) polymerase; c, cleaved; Co. si, control siRNA; Fo. si, FOXO3 siRNA.

    Journal: Oncology Letters

    Article Title: Mitoxantrone induces apoptosis in osteosarcoma cells through regulation of the Akt/FOXO3 pathway

    doi: 10.3892/ol.2018.8547

    Figure Lengend Snippet: FOXO3 dependent-apoptosis in U2OS cells treated with MTZ. Western blotting results of U2OS cells transfected with control or FOXO3 siRNA and treated with MTZ (0 and 0.5 µM) for 48 h, followed by blotting with specific antibodies as indicated. Graphs with quantitative data were based on the relative ratio of the indicated protein to β-actin. MTZ, mitoxantrone; EL, extra-large; FOXO3, forkhead-box O3; PARP1, poly(ADP-ribose) polymerase; c, cleaved; Co. si, control siRNA; Fo. si, FOXO3 siRNA.

    Article Snippet: U2OS cells [2×104 /well in 100 µl DMEM (Gibco; Thermo Fisher Scientific, Inc.)] were seeded into 96-well plates and incubated at 37°C for 18 h in a CO2 incubator.

    Techniques: Western Blot, Transfection

    Inhibition of pS473 Akt and translocation of FOXO3 protein from the cytoplasm to the nucleus in osteosarcoma cells treated with MTZ. (A) Nuclear fraction western blot analysis results of U2OS cells treated with MTZ (0, 0.25, 0.5 and 1 µM). The levels of the indicated proteins in the cytoplasm and nucleus were analyzed by western blotting using specific antibodies. GAPDH and Lamin B1 were as loading controls. (B) Confocal microscopy results of U2OS and MG63 cells treated with MTZ (1 µM) for 2 h. Following counterstaining with DAPI to indicate the nuclei, fluorescence images were captured with a confocal microscope. Scale bar, 10 µm. Cyto, cytoplasm; nucl, nuclei; FOXO3, forkhead-box O3; MTZ, mitoxantrone.

    Journal: Oncology Letters

    Article Title: Mitoxantrone induces apoptosis in osteosarcoma cells through regulation of the Akt/FOXO3 pathway

    doi: 10.3892/ol.2018.8547

    Figure Lengend Snippet: Inhibition of pS473 Akt and translocation of FOXO3 protein from the cytoplasm to the nucleus in osteosarcoma cells treated with MTZ. (A) Nuclear fraction western blot analysis results of U2OS cells treated with MTZ (0, 0.25, 0.5 and 1 µM). The levels of the indicated proteins in the cytoplasm and nucleus were analyzed by western blotting using specific antibodies. GAPDH and Lamin B1 were as loading controls. (B) Confocal microscopy results of U2OS and MG63 cells treated with MTZ (1 µM) for 2 h. Following counterstaining with DAPI to indicate the nuclei, fluorescence images were captured with a confocal microscope. Scale bar, 10 µm. Cyto, cytoplasm; nucl, nuclei; FOXO3, forkhead-box O3; MTZ, mitoxantrone.

    Article Snippet: U2OS cells [2×104 /well in 100 µl DMEM (Gibco; Thermo Fisher Scientific, Inc.)] were seeded into 96-well plates and incubated at 37°C for 18 h in a CO2 incubator.

    Techniques: Inhibition, Translocation Assay, Western Blot, Confocal Microscopy, Fluorescence, Microscopy

    A3H haplotype I has greater nuclear localization than haplotype II. ( a ) Representative images of A3H-I (untagged), A3H-II (untagged), A3B-HA and A3G-HA in SK-BR-3, HeLa and U2OS cells. The 20 μm scale applies to all images. ( b ) Whisker plots quantifying the subcellular localization data as nuclear-to-cytoplasmic ratios for n > 50 cells per condition. The average is shown, the error box represents the first and third quartiles, and the whiskers extend to the highest value within 1.5 × the interquartile range ( P values determined by two-tailed Welch's t -test).

    Journal: Nature Communications

    Article Title: The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis

    doi: 10.1038/ncomms12918

    Figure Lengend Snippet: A3H haplotype I has greater nuclear localization than haplotype II. ( a ) Representative images of A3H-I (untagged), A3H-II (untagged), A3B-HA and A3G-HA in SK-BR-3, HeLa and U2OS cells. The 20 μm scale applies to all images. ( b ) Whisker plots quantifying the subcellular localization data as nuclear-to-cytoplasmic ratios for n > 50 cells per condition. The average is shown, the error box represents the first and third quartiles, and the whiskers extend to the highest value within 1.5 × the interquartile range ( P values determined by two-tailed Welch's t -test).

    Article Snippet: HeLa ce lls were cultured in DMEM (HyClone) and SK-BR-3 and U2OS cells in McCoy's 5A (Corning), each supplemented with 10% foetal bovine serum and 0.5% Pen/Strep.

    Techniques: Whisker Assay, Two Tailed Test

    DAMP release from tumor cells treated with DTT-205 and DTT-304. The exposure of calreticulin (CALR) in human osteosarcoma U2OS cells was measured by flow cytometry using polyclonal anti-CALR antibody while excluding cells that lost cytoplasmic membrane integrity and thus incorporated the exclusion dye propidium iodide (PI) ( a, b ). Exodus of high mobility group box 1 (HMGB1) from the cells into cell culture supernatants was monitored by HMGB1-specific enzyme-linked immunosorbent assay ELISA. Absorbance was measured and concentrations were calculated based on standards ( c, d ). Both CALR and HMGB1 were emitted by both DTT-205 and DTT-304 in a dose-dependent fashion. The production of type I interferon (IFN) was measured in by reverse transcription quantitative real time polymerase chain reaction qRT-qPCR from purified mRNA of cells treated with DTT compounds ( e, f ) (mean ± SD of triplicate assessments; Student’s t test, * p

    Journal: Cell Death & Disease

    Article Title: Oncolysis with DTT-205 and DTT-304 generates immunological memory in cured animals

    doi: 10.1038/s41419-018-1127-3

    Figure Lengend Snippet: DAMP release from tumor cells treated with DTT-205 and DTT-304. The exposure of calreticulin (CALR) in human osteosarcoma U2OS cells was measured by flow cytometry using polyclonal anti-CALR antibody while excluding cells that lost cytoplasmic membrane integrity and thus incorporated the exclusion dye propidium iodide (PI) ( a, b ). Exodus of high mobility group box 1 (HMGB1) from the cells into cell culture supernatants was monitored by HMGB1-specific enzyme-linked immunosorbent assay ELISA. Absorbance was measured and concentrations were calculated based on standards ( c, d ). Both CALR and HMGB1 were emitted by both DTT-205 and DTT-304 in a dose-dependent fashion. The production of type I interferon (IFN) was measured in by reverse transcription quantitative real time polymerase chain reaction qRT-qPCR from purified mRNA of cells treated with DTT compounds ( e, f ) (mean ± SD of triplicate assessments; Student’s t test, * p

    Article Snippet: High-throughput assessment of cell death In total, 5 x 103 U2OS cells were seeded into black 96-well µclear imaging plates (Greiner Bio-One) and allowed to adapt for 24 h. Thereafter the cells were treated with the DTT compounds and respective controls and incubated for additional 6 or 24 h before either 1 µM of DAPI or a mixture of 1 µM Hoechst, and 1 µM propidium iodide were added immediately before monitoring the uptake of the exclusion dye in a minimum of four view fields per well by means of an ImageXpress micro XL automated bioimager (Molecular Devices) equipped with a PlanApo 20 × /0.75 NA objective (Nikon, Tokyo, Japan).

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Purification

    DTT peptides induce the formation of lipid droplets. Human osteosarcoma U2OS cells were treated with 2.5 µM of either DTT-205 or DTT-304 for 6 h. Cells were fixed and subjected to electron microscopy. The overview micrographs in the upper panel depict necrotic morphologies (N) of some of the treated cells and the high magnification micrographs in the lower panel show the formation of lipid droplets (L) in intact cells. Size bars equals 10 µm (upper panel) and 1 µm (lower panel). Representative images ( a ) and quantifications ( b, c ) are depicted (mean ± SD of a minimum of five view fields). The formation of lipid droplets in response to increasing doses from 0.65 to 10 µM DTT peptides was quantified by means of the lipophilic dye Nile Red at 6 h and 24 h post treatment in epifluorescence microscopy. Representative images ( d ) and quantifications ( e, f ) are shown (mean ± SD of triplicate assessments, Student’s t test, p

    Journal: Cell Death & Disease

    Article Title: Oncolysis with DTT-205 and DTT-304 generates immunological memory in cured animals

    doi: 10.1038/s41419-018-1127-3

    Figure Lengend Snippet: DTT peptides induce the formation of lipid droplets. Human osteosarcoma U2OS cells were treated with 2.5 µM of either DTT-205 or DTT-304 for 6 h. Cells were fixed and subjected to electron microscopy. The overview micrographs in the upper panel depict necrotic morphologies (N) of some of the treated cells and the high magnification micrographs in the lower panel show the formation of lipid droplets (L) in intact cells. Size bars equals 10 µm (upper panel) and 1 µm (lower panel). Representative images ( a ) and quantifications ( b, c ) are depicted (mean ± SD of a minimum of five view fields). The formation of lipid droplets in response to increasing doses from 0.65 to 10 µM DTT peptides was quantified by means of the lipophilic dye Nile Red at 6 h and 24 h post treatment in epifluorescence microscopy. Representative images ( d ) and quantifications ( e, f ) are shown (mean ± SD of triplicate assessments, Student’s t test, p

    Article Snippet: High-throughput assessment of cell death In total, 5 x 103 U2OS cells were seeded into black 96-well µclear imaging plates (Greiner Bio-One) and allowed to adapt for 24 h. Thereafter the cells were treated with the DTT compounds and respective controls and incubated for additional 6 or 24 h before either 1 µM of DAPI or a mixture of 1 µM Hoechst, and 1 µM propidium iodide were added immediately before monitoring the uptake of the exclusion dye in a minimum of four view fields per well by means of an ImageXpress micro XL automated bioimager (Molecular Devices) equipped with a PlanApo 20 × /0.75 NA objective (Nikon, Tokyo, Japan).

    Techniques: Electron Microscopy, Epifluorescence Microscopy

    Organellar targeting of DTT-205 and DTT-304. Human osteosarcoma U2OS cells stably expressing the nuclear marker histone H2B together with red fluorescent protein (RFP), the ER marker calreticulin (CALR) labeled with green fluorescent protein (GFP), galactose-1-phosphate uridylyltransferase GALT1, a marker of the Golgi apparatus fused to GFP, DIABLO co-expressing GFP as an indicator for mitochondria and LAMP1-GFP as a lysosomal marker were treated with 1.25 µM Pacific blue-labeled DTT peptides in the presence or absence of lysosomal acidification that was blocked or not with bafilomycin A1 (BAFA1). Both DTT-205 and DTT-304 accumulated in lysosomal structures, an effect that was inhibited with BAFA1. Representative images of confocal assessment ( a, b ; size bar equals 5 µm) and relative cooccurrence of Pacific Blue label with organellar markers was assessed ( c, d ; mean ± SEM of a minimum of five view fields). Wild-type U2OS cells were stained with LysoTracker green and the decrease in lysosomal content was assessed upon treatment 0.65 or 1.25 µM DTT peptides for 6 h by epifluorescence microscopy. Representative images ( e ) and quantifications ( f, g ) are depicted (size bar equals 10 µm; mean ± SD of triplicate assessments, Student’s t test, * p

    Journal: Cell Death & Disease

    Article Title: Oncolysis with DTT-205 and DTT-304 generates immunological memory in cured animals

    doi: 10.1038/s41419-018-1127-3

    Figure Lengend Snippet: Organellar targeting of DTT-205 and DTT-304. Human osteosarcoma U2OS cells stably expressing the nuclear marker histone H2B together with red fluorescent protein (RFP), the ER marker calreticulin (CALR) labeled with green fluorescent protein (GFP), galactose-1-phosphate uridylyltransferase GALT1, a marker of the Golgi apparatus fused to GFP, DIABLO co-expressing GFP as an indicator for mitochondria and LAMP1-GFP as a lysosomal marker were treated with 1.25 µM Pacific blue-labeled DTT peptides in the presence or absence of lysosomal acidification that was blocked or not with bafilomycin A1 (BAFA1). Both DTT-205 and DTT-304 accumulated in lysosomal structures, an effect that was inhibited with BAFA1. Representative images of confocal assessment ( a, b ; size bar equals 5 µm) and relative cooccurrence of Pacific Blue label with organellar markers was assessed ( c, d ; mean ± SEM of a minimum of five view fields). Wild-type U2OS cells were stained with LysoTracker green and the decrease in lysosomal content was assessed upon treatment 0.65 or 1.25 µM DTT peptides for 6 h by epifluorescence microscopy. Representative images ( e ) and quantifications ( f, g ) are depicted (size bar equals 10 µm; mean ± SD of triplicate assessments, Student’s t test, * p

    Article Snippet: High-throughput assessment of cell death In total, 5 x 103 U2OS cells were seeded into black 96-well µclear imaging plates (Greiner Bio-One) and allowed to adapt for 24 h. Thereafter the cells were treated with the DTT compounds and respective controls and incubated for additional 6 or 24 h before either 1 µM of DAPI or a mixture of 1 µM Hoechst, and 1 µM propidium iodide were added immediately before monitoring the uptake of the exclusion dye in a minimum of four view fields per well by means of an ImageXpress micro XL automated bioimager (Molecular Devices) equipped with a PlanApo 20 × /0.75 NA objective (Nikon, Tokyo, Japan).

    Techniques: Stable Transfection, Expressing, Marker, Labeling, Staining, Epifluorescence Microscopy

    Cell death induced by DTT-205 and DTT-304. Human osteosarcoma U2OS cells were treated with 0.65 to 10 µM DTT peptides for the indicated time and following assessed for the activation of caspase-3 (CASP3). The pan-kinase inhibitor staurosporine (STS) was used as positive control. Representative images ( a, b ) and quantifications of CASP3 activation ( c, d ) are depicted while pyknosis was assessed by measuring the decrease in Hoechst 33342-stained nuclear area ( e, f ) by conventional microscopy (size bar equals 10 µm; mean ± SD of triplicate assessments, Student’s t test, * p

    Journal: Cell Death & Disease

    Article Title: Oncolysis with DTT-205 and DTT-304 generates immunological memory in cured animals

    doi: 10.1038/s41419-018-1127-3

    Figure Lengend Snippet: Cell death induced by DTT-205 and DTT-304. Human osteosarcoma U2OS cells were treated with 0.65 to 10 µM DTT peptides for the indicated time and following assessed for the activation of caspase-3 (CASP3). The pan-kinase inhibitor staurosporine (STS) was used as positive control. Representative images ( a, b ) and quantifications of CASP3 activation ( c, d ) are depicted while pyknosis was assessed by measuring the decrease in Hoechst 33342-stained nuclear area ( e, f ) by conventional microscopy (size bar equals 10 µm; mean ± SD of triplicate assessments, Student’s t test, * p

    Article Snippet: High-throughput assessment of cell death In total, 5 x 103 U2OS cells were seeded into black 96-well µclear imaging plates (Greiner Bio-One) and allowed to adapt for 24 h. Thereafter the cells were treated with the DTT compounds and respective controls and incubated for additional 6 or 24 h before either 1 µM of DAPI or a mixture of 1 µM Hoechst, and 1 µM propidium iodide were added immediately before monitoring the uptake of the exclusion dye in a minimum of four view fields per well by means of an ImageXpress micro XL automated bioimager (Molecular Devices) equipped with a PlanApo 20 × /0.75 NA objective (Nikon, Tokyo, Japan).

    Techniques: Activation Assay, Positive Control, Staining, Microscopy

    Effects of CX-4945 and TDB on clone formation and survival. 100 U2OS cells were plated for each well, treated for 24 h with the indicated inhibitors, and then grown for further 6 days in the absence of the inhibitors. Two representative images are shown for each condition (2.5x objective, magnification-changer 1.6); at least two separate experiments in triplicate were performed.

    Journal: BioMed Research International

    Article Title: Different Persistence of the Cellular Effects Promoted by Protein Kinase CK2 Inhibitors CX-4945 and TDB

    doi: 10.1155/2015/185736

    Figure Lengend Snippet: Effects of CX-4945 and TDB on clone formation and survival. 100 U2OS cells were plated for each well, treated for 24 h with the indicated inhibitors, and then grown for further 6 days in the absence of the inhibitors. Two representative images are shown for each condition (2.5x objective, magnification-changer 1.6); at least two separate experiments in triplicate were performed.

    Article Snippet: U2OS cells were seeded in complete culture medium at a density of 3 × 104 cells on each side of the Ibidi culture-insert, into a 24-well plate.

    Techniques:

    Effects of CX-4945 and TDB on spheroid formation. U2OS cells were pretreated for 24 h with the indicated concentrations of inhibitors and then allowed to form spheroids. Images were taken after 24 h and 96 h. Two separate experiments were performed, with triplicates of each condition (2.5x objective, magnification-changer 1.6). Two representative images for each condition are shown.

    Journal: BioMed Research International

    Article Title: Different Persistence of the Cellular Effects Promoted by Protein Kinase CK2 Inhibitors CX-4945 and TDB

    doi: 10.1155/2015/185736

    Figure Lengend Snippet: Effects of CX-4945 and TDB on spheroid formation. U2OS cells were pretreated for 24 h with the indicated concentrations of inhibitors and then allowed to form spheroids. Images were taken after 24 h and 96 h. Two separate experiments were performed, with triplicates of each condition (2.5x objective, magnification-changer 1.6). Two representative images for each condition are shown.

    Article Snippet: U2OS cells were seeded in complete culture medium at a density of 3 × 104 cells on each side of the Ibidi culture-insert, into a 24-well plate.

    Techniques:

    Effects of CX-4945 and TDB on cell migration. Cell migration was assessed by wound-healing assay, seeding U2OS cells in wells containing an insert, and incubating them for 24 h in the presence of the indicated inhibitor (10 μ M) or vehicle. Then the insert was removed ( t = 0) and cells were left to migrate for 48 h, always in the presence of the inhibitors (upper part of the figure, “inhibitors maintained”) or in their absence (lower part of the figure, “inhibitors removed”). The experiment was performed three times, with each condition in duplicate. Representative images at t = 0, t = 24 h, and t = 48 h are shown.

    Journal: BioMed Research International

    Article Title: Different Persistence of the Cellular Effects Promoted by Protein Kinase CK2 Inhibitors CX-4945 and TDB

    doi: 10.1155/2015/185736

    Figure Lengend Snippet: Effects of CX-4945 and TDB on cell migration. Cell migration was assessed by wound-healing assay, seeding U2OS cells in wells containing an insert, and incubating them for 24 h in the presence of the indicated inhibitor (10 μ M) or vehicle. Then the insert was removed ( t = 0) and cells were left to migrate for 48 h, always in the presence of the inhibitors (upper part of the figure, “inhibitors maintained”) or in their absence (lower part of the figure, “inhibitors removed”). The experiment was performed three times, with each condition in duplicate. Representative images at t = 0, t = 24 h, and t = 48 h are shown.

    Article Snippet: U2OS cells were seeded in complete culture medium at a density of 3 × 104 cells on each side of the Ibidi culture-insert, into a 24-well plate.

    Techniques: Migration, Wound Healing Assay

    Comparison of CX-4945 and TDB efficacy on cellular CK2 activity. CEM or U2OS cells were treated for 4 h with increasing concentrations of TDB or CX-4945. Then cells were lysed and CK2 activity was assessed from 1-2 μ g of lysate proteins towards a synthetic CK2-specific peptide. Activity is shown as percent of control vehicle-treated cells (mean of two independent experiments ± SEM). In the box, the IC 50 values for CK2 inhibition in the two cell lines are reported, as extrapolated from the curves above; the in vitro IC 50 values for CK2 are also shown for comparison, as reported in [ 12 ] ( a ) (SEM never exceeding 10%) and [ 7 ] ( b ). DC 50 values (concentrations required to induce 50% of cell death in 24 h) are reported as in [ 12 ] ( a ) for HeLa cells, where a direct comparison between the two compounds in inducing cell death is presented. Statistical significance was calculated using unpaired t -test between control and treated cells ( ∗∗∗∗ p ≤ 0.0001; ∗∗∗ p ≤ 0.001; ∗∗ p ≤ 0.01; ∗ p ≤ 0.05).

    Journal: BioMed Research International

    Article Title: Different Persistence of the Cellular Effects Promoted by Protein Kinase CK2 Inhibitors CX-4945 and TDB

    doi: 10.1155/2015/185736

    Figure Lengend Snippet: Comparison of CX-4945 and TDB efficacy on cellular CK2 activity. CEM or U2OS cells were treated for 4 h with increasing concentrations of TDB or CX-4945. Then cells were lysed and CK2 activity was assessed from 1-2 μ g of lysate proteins towards a synthetic CK2-specific peptide. Activity is shown as percent of control vehicle-treated cells (mean of two independent experiments ± SEM). In the box, the IC 50 values for CK2 inhibition in the two cell lines are reported, as extrapolated from the curves above; the in vitro IC 50 values for CK2 are also shown for comparison, as reported in [ 12 ] ( a ) (SEM never exceeding 10%) and [ 7 ] ( b ). DC 50 values (concentrations required to induce 50% of cell death in 24 h) are reported as in [ 12 ] ( a ) for HeLa cells, where a direct comparison between the two compounds in inducing cell death is presented. Statistical significance was calculated using unpaired t -test between control and treated cells ( ∗∗∗∗ p ≤ 0.0001; ∗∗∗ p ≤ 0.001; ∗∗ p ≤ 0.01; ∗ p ≤ 0.05).

    Article Snippet: U2OS cells were seeded in complete culture medium at a density of 3 × 104 cells on each side of the Ibidi culture-insert, into a 24-well plate.

    Techniques: Activity Assay, Inhibition, In Vitro

    Duration of CK2 inhibition after TDB or CX-4945 removal from treated cells. CEM or U2OS cells were treated with TDB or CX-4945, as indicated, for 24 h. Then cells were washed and the culture medium was replaced with a fresh one devoid of inhibitors. (a) Cells were cultured for a further time, as indicated, and then lysed. CK2 activity was assessed from 1-2 μ g of lysate proteins towards a synthetic CK2-specific peptide. The inhibitor concentration was 3 μ M. Activity is shown as percent of control vehicle-treated cells (mean of three independent experiments ± SEM; statistical significance was calculated using unpaired t -test between control and treated cells ( ∗∗∗∗ p ≤ 0.0001; ∗∗∗ p ≤ 0.001; ∗∗ p ≤ 0.01; ∗ p ≤ 0.05)). (b) Cells were lysed after the first 24 h of culture in the presence of 10 μ M inhibitors (upper blots) or cultured for further 24 h after the inhibitor removal (lower blots). 10 μ g of lysate proteins was analyzed by WB with the indicated antibodies. A quantification of the pSer129 (mean of three independent experiments ± SEM) normalized to Akt1 total amount is shown in the histograms on the right, where 100% values have been assigned to untreated cells of each experimental protocol.

    Journal: BioMed Research International

    Article Title: Different Persistence of the Cellular Effects Promoted by Protein Kinase CK2 Inhibitors CX-4945 and TDB

    doi: 10.1155/2015/185736

    Figure Lengend Snippet: Duration of CK2 inhibition after TDB or CX-4945 removal from treated cells. CEM or U2OS cells were treated with TDB or CX-4945, as indicated, for 24 h. Then cells were washed and the culture medium was replaced with a fresh one devoid of inhibitors. (a) Cells were cultured for a further time, as indicated, and then lysed. CK2 activity was assessed from 1-2 μ g of lysate proteins towards a synthetic CK2-specific peptide. The inhibitor concentration was 3 μ M. Activity is shown as percent of control vehicle-treated cells (mean of three independent experiments ± SEM; statistical significance was calculated using unpaired t -test between control and treated cells ( ∗∗∗∗ p ≤ 0.0001; ∗∗∗ p ≤ 0.001; ∗∗ p ≤ 0.01; ∗ p ≤ 0.05)). (b) Cells were lysed after the first 24 h of culture in the presence of 10 μ M inhibitors (upper blots) or cultured for further 24 h after the inhibitor removal (lower blots). 10 μ g of lysate proteins was analyzed by WB with the indicated antibodies. A quantification of the pSer129 (mean of three independent experiments ± SEM) normalized to Akt1 total amount is shown in the histograms on the right, where 100% values have been assigned to untreated cells of each experimental protocol.

    Article Snippet: U2OS cells were seeded in complete culture medium at a density of 3 × 104 cells on each side of the Ibidi culture-insert, into a 24-well plate.

    Techniques: Inhibition, Cell Culture, Activity Assay, Concentration Assay, Western Blot