u2os human bone sarcoma cell line  (ATCC)

Journal: bioRxiv

Article Title: Integrating Cell Painting and Thermal Proteome Profiling for Inference of Targets and Mechanism of Action

doi: 10.1101/2025.05.30.657006

Figure Lengend Snippet: A.Lollipop chart for subcellular location of the 67 versus 40 proteins found to be either stabilized or destabilized in TPP for compounds (+)-JQ1 (top) and I-BET151 (middle), respectively. Subcellular location of each protein, based on immunohistochemistry and confocal microscopy, was retrieved from the Human Protein Atlas ( proteinatlas.org ). When a protein had more than one subcellular location assigned, that protein was included once per subcellular location in the graph. Bottom graph show a Venn diagram of the overlap between proteins stabilized/destabilized in (+)-JQ1 and I-BET151. Only 11 proteins were found to overlap between the two compounds. B. Physical protein-protein interaction (PPI) networks for the proteins found to be either stabilized or destabilized in TPP for compounds (+)-JQ1 and I-BET151, respectively. PPI networks were retrieved from the STRING db. Nodes are colored by subcellular location (from Human Protein Atlas). Large nodes correspond to proteins detected by TPP, where small nodes are additionally added nodes from the STRING db during network retrieval. C. Top: Physical PPI networks for (+)-JQ1 (same as B), except green nodes here indicate proteins found to be stabilized by (+)-JQ1 in cell extract. Bottom: Venn diagram for overlap of proteins detected in whole cells versus cell extract after perturbation with (+)-JQ1. D. Radar chart for features in cell painting data for (+)-JQ1 and I-BET151. Features were grouped into categories based on two criteria: (i) Cell Profiler module, i.e. Intensity (I), Correlation (C), Granularity (G), Location (L) and RadialDistribution (RD); and (ii) stains, i.e. Nucleus (Hoechst), ER (Concanavalin A), Nucleoli and cytoplasmic RNA (SYTO14), Golgi apparatus and F-actin cytoskeleton (WGA and Phalloidin) and Mitochondria (Mitotracker). Features were only consider for the object Cell, except for features from the stain for Nucleus, which was only considered in the object Nucleus. Additionally, area-shape related features were grouped by cell compartment, i.e. Cell (C), Cytoplasm (Cy) and Nucleus (N). E. t-SNE for the morphological features in the SPECS cell painting data on U2OS cells. Blue dots show the location of all cells treated with BET bromodomain inhibitors sharing at least one target with either (+)-JQ1 or I-BET151. Despite several BET bromodomain inhibitors clustering close to (+)-JQ1 or I-BET151, there are also several compounds with distinctly different morphological changes.

Article Snippet: For the purpose of this study, U2OS human bone sarcoma cell line (sourced from ATCC U2OS #HTB-96) was expanded and painted at passage number +8.

Techniques: Immunohistochemistry, Confocal Microscopy, Staining

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: UFL1 and C53 interact with γTuRC proteins. ( A ) Immunoprecipitation experiments. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized Abs specific to UFL1 301–389 , C53, γ-tubulin (γ-Tb), or GCP2. The blots were probed with Abs to UFL1, C53, γ-tubulin (γ-Tb), GCP2, GCP4, or calcineurin (Calcin.; negative control). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carriers incubated with cell extracts ( lane 4 ). ( B ) Isotype controls. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized rabbit Ab to myosin or mouse mAb to MAP2 (IgG2b). Blots were probed with Abs to UFL1, C53, γ-tubulin (γ-Tb), GCP2, or GCP4. Load ( lane 1 ), immobilized Abs not incubated with cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and carriers without Abs incubated with cell extracts ( lane 4 ). ( C ) The size distribution of UFL1, C53, γ-tubulin (γ-Tb), GCP2, and actin in U2OS whole-cell extracts fractionated on the Superose 6 column. The calibration standards (in kDa) are indicated on the top. The numbers at the bottom denote individual fractions. ( D ) Pooled fractions (Nos. 15–16) and (Nos. 23–24) from fractionation shown in panel ( C ) were precipitated with Ab to γ-tubulin. The blots were probed with Abs to UFL1, C53, and γ-tubulin (γ-Tb). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carriers incubated with cell extracts ( lane 4 ).

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Immunoprecipitation, Negative Control, Incubation, Fractionation

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Subcellular localization of UFL1 and C53. ( A ) U2OS cells expressing TagRFP-tagged proteins were fixed and stained with Ab to γ-tubulin. Localization of C53-TagRFP ( a , d ) and γ-tubulin ( b , e ). Superposition of images ( c , f ) C53-TagRFP, red; γ-tubulin, green; DAPI, blue). Localization of UFL1-TagRFP ( g ) and γ-tubulin ( h ). Superposition of images ( i , UFL1-TagRFP, red; γ-tubulin, green; DAPI, blue). Arrows indicate the same positions. Fixation D/F/M. Scale bar, 20 μm ( a – c , g – i ), 5 μm ( d – f ). ( B ) Association of proteins with purified centrosomes. Centrosomes enriched by centrifugation onto a Ficoll cushion were further purified by sucrose gradient centrifugation. The gradient was fractionated from the bottom. Individual fractions are indicated on the top, sucrose density in the fractions is shown at the bottom. Blots were probed with Abs to pericentrin, CDK5RAP2, ODF2, γ-tubulin (γ-Tb), C53, UFL1, PKCα, histone H1.4, and calnexin.

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Staining, Purification, Centrifugation, Gradient Centrifugation

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Deletion of UFL1 or C53 induces UPR and expansion of ER. ( A ) Immunoblot analysis of calnexin and PDI in whole-cell lysates of U2OS cells lacking UFL1 or C53. GAPDH served as a loading control. Densitometric quantification of immunoblots is shown on the right. Relative intensities of corresponding proteins normalized to control cells and the amount of GAPDH in individual samples. Values indicate mean ± SD ( n = 4 for calnexin; n = 3 for PDI). ( B ) Immunofluorescence microscopy. ( a – d ) Control U2OS cells, ( e – h ) UFL1-deficient cells (UFL1_KO) and ( i – l ) C53-deficient cells (C53_KO). Cells were fixed and double-labeled for calnexin ( a , e , i ) and β-tubulin ( b , f , j ; Microtubules). Higher magnification views of the regions delimited by rectangles are shown on the right of images from control ( c , d ), UFL1_KO ( g , h ), and C53_KO ( k , l ) cells. The images ( a , e , i ) and ( c , g , k ) were collected and processed in the same manner. Fixation F/Tx. Scale bars, 20 μm ( j ), and 5 µm ( l ). ( C ) ER area quantification in fixed cells stained with Ab to calnexin. ER area coefficients (area occupied by ER/area free of ER) were calculated from fluorescence intensities for calnexin. The distributions of ER area coefficients (arbitrary units [AU]) are shown as box plots (four independent experiments, ≥16 cells counted for each experimental condition). Box plot of area coefficients in UFL1_KO ( n = 111) and C53_KO cells ( n = 127) relative to control cells ( n = 149). The bottom and top of the box represent the 25th and 75th percentiles. Whiskers below and above the box indicate the 10th and 90th percentiles. ( A , C ) One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine statistical significance. *, p < 0.05, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control, Immunofluorescence, Microscopy, Labeling, Staining, Fluorescence

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Generation of ER stress by tunicamycin increases centrosomal microtubule nucleation. U2OS cells were treated with 1 µg/mL tunicamycin (+Tunicam.) or DMSO carrier (Control) for 24 h. ( A ). Effect of tunicamycin on ER expansion and subcellular distribution of calnexin, PDI, and ER stress-induced transcription factor DDIT3. ( a , b ) Visualization of ER in live cells by ER-Tracker. ( c – h ) Fixed cells stained for calnexin ( c , d ), PDI ( e , f ), and DDIT3 ( g , h ). Insets represent an enlargement of the boxed area. Fixation F/Tx. Pairs of images ( a , b ), ( c , d ), ( e , f ), and ( g , h ) were collected and processed in the same manner. Scale bars, 20 μm ( h ) and 5 µm (inset in f ). ( B ) ER area quantification in live cells stained with ER-Tracker. The distributions of ER area coefficients (area occupied by ER/area free of ER; arbitrary units [AU]) are shown as box plots (three independent experiments, ≥23 cells counted for each experimental condition). Box plot of ER area coefficients in tunicamycin-treated cells ( n = 81) relative to control cells ( n = 108). ( C ) ER area quantification in fixed cells stained with Ab to calnexin. The distributions of ER area coefficients (area occupied by ER/area free of ER; arbitrary units [AU]) are shown as box plots (three independent experiments, ≥19 cells counted for each experimental condition). Box plot of ER area coefficients in tunicamycin-treated cells ( n = 80) relative to control cells ( n = 65). ( D , E ) The distributions of α-tubulin or γ-tubulin fluorescence intensities (arbitrary units [AU]) in 2-μm ROIs at 3.0 min of microtubule regrowth in control and tunicamycin-treated cells are shown as box plots (four independent experiments, >27 cells counted for each experimental condition). Box plot of α-tubulin ( D ) and γ-tubulin ( E ) fluorescence intensities in tunicamycin-treated cells ( n = 234) relative to control cells ( n = 181). ( F ) Time-lapse imaging of control and tunicamycin-treated cells expressing EB3-mNeonGreen. Still images of EB3 (Single frame) and tracks of EB3 comets over 10 s (10 frames project.). Scale bar, 5 µm. ( G ) Microtubule nucleation rate (EB3 comets/min) in tunicamycin-treated cells relative to control cells. Three independent experiments (at least 9 cells counted in each experiment). Control ( n = 31), tunicamycin-treated cells ( n = 31). The bold and thin lines within the dot plot represent mean ± SD. ( B – E ) The bold and thin lines within the box represent mean and median (the 50th percentile), respectively. The bottom and top of the box represent the 25th and 75th percentiles. Whiskers below and above the box indicate the 10th and 90th percentiles. ( B – E , G ) Two-tailed, unpaired Student’s t -test was performed to determine statistical significance. **** p < 0.0001.

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Control, Staining, Fluorescence, Imaging, Expressing, Two Tailed Test

Journal: Cancer research

Article Title: FOXP3 Regulates Sensitivity of Cancer Cells to Irradiation by Transcriptional Repression of BRCA1

doi: 10.1158/0008-5472.CAN-12-2481

Figure Lengend Snippet: FOXP3 represses DNA repairs. A. Representative histograms depicting distribution of GFP signals. U2OS cells with integrated DR-GFP reporter were stably transduced with scrambled or FOXP3 shRNA lentiviral vectors. Then the cells were infected with adenovirus carried restriction enzyme I-Sce I. Since the HR DNA repair corrects the GFP open-reading frame, the repair efficiency was measured by GFP+ cells recorded with flow cytometry.. B. Upper panel shows summary data on DNA repairing efficiency as measured by GFP+ cells. Data shown are means +/− S.D. from 3 independent experiments. *** P < 0.001. Lower panel shows FOXP3 protein expressions detected by Western blot, as shown in the lower panel. C. Comet assay of DNA damage response. U2OS cells stably transduced with scramble or FOXP3 shRNA were irradiated for 60GY. The cells were cultured for the given periods and then subject to agarose gel electrophoresis. The photographs show the momentum of the nuclear DNA movement. D. Summary data of momentum of cellular DNA. Data shown are means +/− S.D. from three independent experiments. *** P< 0.001.

Article Snippet: Breast cancer cell line MCF-7 and immortalized mammary epithelial cell line MCF-10A, human sarcoma cell line U2OS, human colon cancer cell line HCT116 and mouse mammary tumor cell line 4T1 were purchased from the American Type Culture Collection, none of the cells have been in tissue culture for more than six month prior to use.

Techniques: Stable Transfection, Transduction, shRNA, Infection, Flow Cytometry, Western Blot, Single Cell Gel Electrophoresis, Irradiation, Cell Culture, Agarose Gel Electrophoresis

Journal: Cancer research

Article Title: FOXP3 Regulates Sensitivity of Cancer Cells to Irradiation by Transcriptional Repression of BRCA1

doi: 10.1158/0008-5472.CAN-12-2481

Figure Lengend Snippet: FOXP3-BRCA1 interaction regulates DNA repair in U2OS cells. A, B. U2OS cells integrated with DR-GFP reporter were infected either with scrambled or FOXP3 shRNA. Transfectants were selected by puromycin for two weeks. The U2OS cells with scramble and FOXP3 shRNA were treated with scrambled RNAi (S) or BRCA1 RNAi. A. FOXP3 and BRCA1 expression. B. Repair of DSB as measured by GFP positive cells with flow cytometry. Data shown in B are means +/− S.D. from 3 independent experiments. C. Comet assay to measure repair of γ-ray-induced DNA damages. U2OS cells with scramble and FOXP3 shRNA, FOXP3 and/or BRCA1 ShRNAs were irradiated for 60 Gy. The cells were cultured for given periods and used for comet assays. Data showed were +/−S.D. means from three independent experiments. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: Breast cancer cell line MCF-7 and immortalized mammary epithelial cell line MCF-10A, human sarcoma cell line U2OS, human colon cancer cell line HCT116 and mouse mammary tumor cell line 4T1 were purchased from the American Type Culture Collection, none of the cells have been in tissue culture for more than six month prior to use.

Techniques: Infection, shRNA, Expressing, Flow Cytometry, Single Cell Gel Electrophoresis, Irradiation, Cell Culture