cell culture ht1080 cells  (ATCC)

Journal: Nucleic acids research

Article Title: Timed chromatin invasion during mitosis governs prototype foamy virus integration site selection and infectivity.

doi: 10.1093/nar/gkaf449

Figure Lengend Snippet: Figure 3. Conserved Gag CBS residues are essential for timely mitotic chromatin capture. Gag localization during PFV infection. G2 / M-phase synchronized HT1080 cells were transduced with PFV particles encoding WT, R540Q, or Y537Q Gag and fixed at different time points after drug release, corresponding to different mitotic phases indicated in orange. Gag proteins were detected using polyclonal anti-PFV Gag antiserum (green); the nuclear en v elope w as stained with anti-Lamin A / C antibodies (red) and cellular DNA with DAPI (gra y). White arro ws sho w MTOC accumulation of PFV Gag. Scale bars: 20 μm. Percentages of chromatin-bound Gag are indicated for each mitotic phase. See “Materials and methods” section for detailed information on quantification. Results are representative of those observed across at least five independent experiments.

Article Snippet: For each time oint measured, data of this second control (binding of Blitz uffer on biosensors loaded with biotinylated Gag WT pepide) were subtracted to every condition and were plotted on urves, using Prism 9 software. ell culture uman fibrosarcoma cell line HT1080 (ATCC CCL-121) and roteoglycan-deficient packaging cell line 293T-25A [ 26 ] were aintained in Dulbecco’s modified Eagle medium (DMEM; isher), supplemented with 50 μg / ml of gentamicin (Fisher cientific) and 10% fetal calf serum (FCS) (Eurobio Scientific). iral vector production FV particles were produced using a four-component system, ased on a protocol described in [ 26 ].

Techniques: Infection, Transduction, Staining

Journal: Nucleic acids research

Article Title: Timed chromatin invasion during mitosis governs prototype foamy virus integration site selection and infectivity.

doi: 10.1093/nar/gkaf449

Figure Lengend Snippet: Figure 4. Conserved PFV Gag CBS residues are required for optimal infectivity. ( A ) Six days post-infection, GFP-positive cells were counted by flow cytometry as relative measures of infectivity. Error bars are SDs determined from at least three independent infections; the WT values in each e xperiment w ere set to 10 0%. ( B ) Quantit ativ e PCR of integrated vDNA, 6 da y s after HT1080 infection, with PFV v ector particles carrying WT, R540Q, or Y537Q Gag with WT IN or WT Gag containing virus with catalytically inert D185N / E221Q IN (IN-NQ). Results are expressed as percentage relative to the WT condition, which was set to 100%. Statistical analyses were performed using the ordinary one-way ANO V A, with Tukey’s multiple comparisons tests (*** P < .0 0 05; **** P < .0 0 01).

Article Snippet: For each time oint measured, data of this second control (binding of Blitz uffer on biosensors loaded with biotinylated Gag WT pepide) were subtracted to every condition and were plotted on urves, using Prism 9 software. ell culture uman fibrosarcoma cell line HT1080 (ATCC CCL-121) and roteoglycan-deficient packaging cell line 293T-25A [ 26 ] were aintained in Dulbecco’s modified Eagle medium (DMEM; isher), supplemented with 50 μg / ml of gentamicin (Fisher cientific) and 10% fetal calf serum (FCS) (Eurobio Scientific). iral vector production FV particles were produced using a four-component system, ased on a protocol described in [ 26 ].

Techniques: Infection, Flow Cytometry, Virus

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: The Histone Deacetylase Inhibitor Vorinostat Selectively Sensitizes Fibrosarcoma Cells to Chemotherapy

doi: 10.1002/jor.21274

Figure Lengend Snippet: Vorinostat inhibits soft tissue sarcoma and normal fibroblast proliferation. (A) Representative phase-contrast photomicrographs (×200) indicating decreased HT1080, SK-LMS-1, SW872 (STS), and WS-1 normal fibroblast proliferation and altered morphology after vorinostat treatment for 72 h. (B) STS and normal fibroblast proliferation were decreased by vorinostat treatment, as determined by counting cells after 24 h (white bars) or 72 h (black bars). Bars represent the mean and whiskers represent the SEM of at least four biological replicates. # and * denote statistical significance versus cells treated with vehicle after 24 or 72 h, respectively (p < 0.05).

Article Snippet: Cell Culture Human HT1080 fibrosarcoma, SK-LMS-1 leiomyosarcoma, and SW872 liposarcoma cells, and WS-1 normal human fibroblasts were obtained from the ATCC (Manassas, VA) and cultured in minimal essential media (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), nonessential amino acids (Gibco), sodium pyruvate (Gibco), and penicillin–streptomycin (Gibco) at 37°C under 5% CO 2 .

Techniques:

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: The Histone Deacetylase Inhibitor Vorinostat Selectively Sensitizes Fibrosarcoma Cells to Chemotherapy

doi: 10.1002/jor.21274

Figure Lengend Snippet: Vorinostat does not affect basal apoptosis levels in soft tissue sarcoma cells and normal fibroblasts. STS cells and normal fibroblasts were treated with 1.0 μM vorinostat or vehicle for 24 or 72 h and then trypsinized, fixed, stained for cleaved caspase 3 (BD Pharmingen™) and analyzed by flow cytometry. (A) Representative histograms of cleaved caspase 3 staining in HT1080 fibrosarcoma cells treated with vehicle or vorinostat. The negative control sample (first row, first histogram) was treated with vehicle for 72 h and then stained with the secondary Alexa Fluor® 647 antibody (Invitrogen) only. Puromycin (first row, second histogram) was used to induce apoptosis as a positive control for cleaved caspase 3 staining. (B) Vorinostat only moderately affects caspase 3 cleavage and then only at the supratherapeutic 3.0 μM dose in all cell lines tested. Bars represent the mean and whiskers represent the SEM of at least four biological replicates. # and * denote statistical significance versus cells treated with vehicle after 24 or 72 h, respectively (p < 0.05).

Article Snippet: Cell Culture Human HT1080 fibrosarcoma, SK-LMS-1 leiomyosarcoma, and SW872 liposarcoma cells, and WS-1 normal human fibroblasts were obtained from the ATCC (Manassas, VA) and cultured in minimal essential media (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), nonessential amino acids (Gibco), sodium pyruvate (Gibco), and penicillin–streptomycin (Gibco) at 37°C under 5% CO 2 .

Techniques: Staining, Flow Cytometry, Negative Control, Positive Control

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: The Histone Deacetylase Inhibitor Vorinostat Selectively Sensitizes Fibrosarcoma Cells to Chemotherapy

doi: 10.1002/jor.21274

Figure Lengend Snippet: Vorinostat sensitizes fibrosarcoma but not normal fibroblasts to doxorubicin. STS cells and normal fibroblasts were treated with 1.0 μM vorinostat or vehicle for 72 h. Doxorubicin was added at the beginning of the third day. (A) Representative (×200) phase-contrast and fluorescent photomicrographs of HT1080 cells plated in chamber slides, treated with vorinostat, doxorubicin, or both and then fixed and stained with anti-cleaved caspase 3 (BD Pharmingen™) and anti-rabbit Alexa Fluor® 488 (Invitrogen) antibodies. I: Phase contrast; II: cleaved caspase 3; III: doxorubicin; IV: DAPI. (B) Vorinostat and doxorubicin synergistically induce apoptosis in HT1080 but not SK-LMS-1 or SW872 STS cells, or WS-1 normal fibroblasts. After a 72-h treatment with vorinostat, doxorubicin, or both, cells were trypsinized, fixed, stained for cleaved caspase 3 and analyzed by flow cytometry. Bars represent the mean and whiskers represent the SEM of at least four biological replicates. Statistical significance is denoted by: * versus vehicle, # versus vorinostat alone, † versus doxorubicin alone.

Article Snippet: Cell Culture Human HT1080 fibrosarcoma, SK-LMS-1 leiomyosarcoma, and SW872 liposarcoma cells, and WS-1 normal human fibroblasts were obtained from the ATCC (Manassas, VA) and cultured in minimal essential media (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), nonessential amino acids (Gibco), sodium pyruvate (Gibco), and penicillin–streptomycin (Gibco) at 37°C under 5% CO 2 .

Techniques: Staining, Flow Cytometry

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: The Histone Deacetylase Inhibitor Vorinostat Selectively Sensitizes Fibrosarcoma Cells to Chemotherapy

doi: 10.1002/jor.21274

Figure Lengend Snippet: Vorinostat inhibits growth and induces chemosensitivity of fibrosarcoma xenografts. Crl:NU-Foxn1nu nude mice were injected subcutaneously with HT1080 cells. One week after inoculation, mice began receiving daily injections of vorinostat or vehicle and weekly injections of doxorubicin or vehicle. (A,B) HT1080 xenograft growth is inhibited by vorinostat alone and further inhibited by the combination of vorinostat and doxorubicin, but not by doxorubicin alone. Data are represented in (A) as the median and interquartile range and in (B) as the minima and maxima (whiskers) and the median, 25th and 75th percentiles (box) at day 21. N = 16 (Veh, VOR) or N = 9 (Veh + Dox, VOR + Dox). (C) Vorinostat treatment induces histone H3 accumulation in xenografts. Statistical significance is denoted by: *p < 0.05 versus vehicle, #p < 0.01 versus vehicle, †p < 0.01 versus doxorubicin alone.

Article Snippet: Cell Culture Human HT1080 fibrosarcoma, SK-LMS-1 leiomyosarcoma, and SW872 liposarcoma cells, and WS-1 normal human fibroblasts were obtained from the ATCC (Manassas, VA) and cultured in minimal essential media (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), nonessential amino acids (Gibco), sodium pyruvate (Gibco), and penicillin–streptomycin (Gibco) at 37°C under 5% CO 2 .

Techniques: Injection

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: The Histone Deacetylase Inhibitor Vorinostat Selectively Sensitizes Fibrosarcoma Cells to Chemotherapy

doi: 10.1002/jor.21274

Figure Lengend Snippet: Combined vorinostat and doxorubicin treatment induces cell death in fibrosarcoma xenografts. (A) Cleaved caspase 3 immunohistochemistry revealed that cell death was greatest in HT1080 xenografts from vorinostat/doxorubicin-treated mice (upper panel). The high magnification inset indicates cleaved caspase 3-positive apoptotic cells in the xenograft from vorinostat/doxorubicin-treated mice. TUNEL staining (middle panel) confirmed that vorinostat/doxorubicin induced more apoptosis in HT1080 compared to vehicle, doxorubicin alone, or vorinostat alone. Nuclei were counterstained using DAPI (lower panel). (B) Quantitation of TUNEL staining in HT1080 xenografts. Statistical significance is denoted by *p < 0.05 versus vehicle, #p < 0.05 versus doxorubicin alone, †p < 0.01 versus vorinostat alone. Scale bar = 50 μm.

Article Snippet: Cell Culture Human HT1080 fibrosarcoma, SK-LMS-1 leiomyosarcoma, and SW872 liposarcoma cells, and WS-1 normal human fibroblasts were obtained from the ATCC (Manassas, VA) and cultured in minimal essential media (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), nonessential amino acids (Gibco), sodium pyruvate (Gibco), and penicillin–streptomycin (Gibco) at 37°C under 5% CO 2 .

Techniques: Immunohistochemistry, TUNEL Assay, Staining, Quantitation Assay