u2os  (ATCC)

Journal: Journal of Cell Science

Article Title: OptoLoop – an optogenetic tool to probe the functional role of genome organization

doi: 10.1242/jcs.264574

Figure Lengend Snippet: Optogenetic manipulation of proximity between repetitive genomic loci. (A) Scheme of OptoLoop consisting of a fusion between dCas9 and the optogenetic protein CRY2. OptoLoop is targeted to specific genomic loci by introducing specific sgRNAs. CRY2–CRY2 interactions activated by blue light bridge targeted loci to form a chromatin loop. (B) Left panel, region of chromosome 19 showing sgIDR3 and sgTCF3 target sites, representative Hi-C contact map (data from ) and BACs used in DNA-FISH to label the IDR3 (magenta) and TCF3 loci (green). Right panel, mCherry channel images of U2OS dCas9–3XmCherry–CRY2 cells transfected with sgIDR3 and sgTCF3, kept in dark or illuminated with blue light for 3 h (1 s pulses every 10 s), and fixed. Scale bars: 5 µm. (C) Left panel, representative image of DNA-FISH for IDR3 and TCF3 with specific BAC FISH probes in U2OS cells. Right panel represents a single cell highlighted in left panel (yellow box); the expansion shows a single allele in this cell. Dashed line denotes the distance between the two FISH signals. Scale bars: 20 µm (left panel), 5 µm (right panel), 1 µm (expansion). (D) IDR3–TCF3 distances, calculated for U2OS dCas9–mCherry–CRY2 polyclonal cells transfected with indicated combinations of sgIDR3 and sgTCF3, kept under dark or illuminated for 3 h (1 s pulses every 10 s). Violin plot corresponds to a representative experiment, with black lines representing median distances. Bar plot represents means of two independent experiments. Each dot represents the median of typically 5000–10,000 alleles analyzed per experiment. (E) Fraction of alleles with IDR3-TCF3 distance <0.27 µm measured from DNA-FISH images for U2OS dCas9–mCherry–CRY2 polyclonal cells and three clones of U2OS dCas9–3XmCherry–CRY2 cells, transfected with indicated combinations of sgIDR3 and sgTCF3, and kept in dark or illuminated for 3 h (1 s pulses every 10 s). Each dot represents the fraction of typically 5000–10,000 alleles analyzed per experiment. Bars represent means of two or three independent experiments. (F) Measurement of cell-to-cell heterogeneity in loop formation. Bars with green shades: observed fraction of cells with none, one or both alleles with IDR3–TCF3 distance <0.27 µm obtained from a representative experiment shown in E with 2500–5000 cells analyzed per sample. Bars with magenta shades: expected fraction of cells with none, one or both alleles with IDR3–TCF3 distance <0.27 µm assuming that alleles from a same cell are independent between each other (Eqn 2). * P <0.05; *** P <0.001; ns, not significant [two-way ANOVAs followed by post-hoc Tukey tests (D,E); paired two-tailed t -test (E); chi-squared test (F)].

Article Snippet: NIH3T3 (mouse fibroblasts, ATCC #CRL-1658), U2OS (from human osteosarcoma, ATCC #HTB-96), HeLa (from human cervical adenocarcinoma, ATCC #CRM-CCL-2) and Lenti-X HEK-293T (from human embryonic kidney, cat. #632180 from Takara Bio, Japan) cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, Waltham, MA, USA) supplemented with 10% (15% for NIH3T3) fetal bovine serum (Gibco, Waltham, MA, USA) plus 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Hi-C, Transfection, Single Cell, Clone Assay, Two Tailed Test

Journal: Journal of Cell Science

Article Title: OptoLoop – an optogenetic tool to probe the functional role of genome organization

doi: 10.1242/jcs.264574

Figure Lengend Snippet: Benchmarking OptoLoop against a previous optogenetic manipulation tool. (A) Scheme of LADL consisting of soluble CRY2wt and a fusion between dCas9 and the CRY2 partner CIBN. dCas9–CIBN tethers specific genomic loci and light-activation induces both CRY2–CRY2 and CRY2–CIBN interactions bridging the targeted loci to form a loop. (B) Images of U2OS cells expressing dCas9–3XGFP–CIBN and mCherry fused to CRY2wt or CRY2olig, transfected with sgIDR3 and sgTCF3, and fixed after being kept under dark or illuminated with blue light pulses for 3 h (1 s pulses every 10 s). White arrows indicate FISH signals corresponding to IDR3–TCF3 loci. Scale bar: 5 µm. (C) Fraction of alleles with IDR3–TCF3 distance <0.27 µm measured from DNA-FISH images of U2OS cell lines stably expressing dCas9-3XGFP-CRY2 and mCherry-CRY2olig (LADL, clone #7) or dCas9-3XmCherry-CRY2 (OptoLoop, clone #3), transfected with sgIDR3 and sgTCF3, and kept under dark or illuminated with blue light for 3 h (1 s pulses every 10 s). Each dot represents the fraction of 5000–7000 alleles analyzed per experiment. Bars represent the means of three independent experiments. * P <0.05; ** P <0.01; *** P <0.001 (two-way ANOVA followed by post-hoc Tukey test).

Article Snippet: NIH3T3 (mouse fibroblasts, ATCC #CRL-1658), U2OS (from human osteosarcoma, ATCC #HTB-96), HeLa (from human cervical adenocarcinoma, ATCC #CRM-CCL-2) and Lenti-X HEK-293T (from human embryonic kidney, cat. #632180 from Takara Bio, Japan) cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, Waltham, MA, USA) supplemented with 10% (15% for NIH3T3) fetal bovine serum (Gibco, Waltham, MA, USA) plus 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Activation Assay, Expressing, Transfection, Stable Transfection

Journal: bioRxiv

Article Title: Constitutive plasma membrane interaction of active Rho GEF Ect2 inhibits cortex contraction pulses

doi: 10.64898/2026.06.03.729549

Figure Lengend Snippet: A) Schematic for domain structures of Ect2 wild-type (Ect2-wt), N-terminally truncated active (ΔN-Ect2) and the catalytically inactive variant with PVQR->AAAA (564-567) substitutions (ΔN-Ect2-DHmut). B) Representative maximum intensity projections of 3D-SIM images of U2OS cells expressing EGFP control or the indicated EGFP-tagged Ect2 constructs. Upper panels show EGFP or EGFP-tagged Ect2 localization, middle panels display F-actin stained with phalloidin, and lower panels present magnified views of the regions depicted in the actin images. Scale bar: 10 µm. n = 7-15 cells. C) Quantification of mean phalloidin fluorescence intensity in wide-field images of cells expressing the indicated constructs. Cells were stained with WGA to visualize cell morphology and with phalloidin to label F-actin. Transfected cells were identified EGFP fluorescence. F-actin intensity was quantified by automated image analysis as described in the Methods. Data represent n ≥ 276 cells from 4 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Bars indicate mean ± SEM. D and H) Representative TIRF images of the Rho activity sensor (mCherry-Rhotekin RBD) co-expressing either EGFP control, constitutively active Ect2 (EGFP-ΔN-Ect2) (D) or constitutively active GEF-H1 C53R (H) . Two phenotypes induced by active Ect2 are shown in (D) : (top) reduced pulsatory Rho sensor signal dynamics and (bottom) peripheral enrichment of Rho sensor signal with slow circumferential movement (white arrow). Frame rate: 3 frames/min, scale bar, 20 µm. E and F) Percent cells with peripheral Rho sensor enrichment (E) and the frequency of Rho sensor pulses in the central cell region (F) ; n ≥ 32 cells from 4 independent experiments. (G) Average pulse frequency of the Rho activity sensor signal upon co-expression of EGFP-control, active EGFP-ΔN-Ect2 or a mutant which cannot bind to active Rho (EGFP-ΔN-Ect2-RBmut); n ≥ 25 cells from 3 independent experiments. Bars indicate mean ± SEM.

Article Snippet: Human U2OS osteosarcoma cells (HTB-96; ATCC) were maintained at 37 °C and 5 % CO 2 humidified atmosphere using standard cell culture techniques (DMEM + GlutaMAXTM medium, 10 % FBS, Life technologies; Gibco).

Techniques: Variant Assay, Expressing, Control, Construct, Staining, Fluorescence, Transfection, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Constitutive plasma membrane interaction of active Rho GEF Ect2 inhibits cortex contraction pulses

doi: 10.64898/2026.06.03.729549

Figure Lengend Snippet: Background-corrected average EGFP intensity in U2OS cells expressing EGFP control (black dots) or EGFP-ΔN-Ect2 (green dots), plotted against the normalized mean pulse frequency of the RBD sensor. Each dot represents a single cell. Data are from n ≥52 cells across three independent experiments.

Article Snippet: Human U2OS osteosarcoma cells (HTB-96; ATCC) were maintained at 37 °C and 5 % CO 2 humidified atmosphere using standard cell culture techniques (DMEM + GlutaMAXTM medium, 10 % FBS, Life technologies; Gibco).

Techniques: Expressing, Control, Single Cell

Journal: bioRxiv

Article Title: Constitutive plasma membrane interaction of active Rho GEF Ect2 inhibits cortex contraction pulses

doi: 10.64898/2026.06.03.729549

Figure Lengend Snippet: A) Representative spinning disk confocal images of fixed U2OS cells transfected with either EGFP control, active Ect2 (EGFP-ΔN-Ect2), or a variant lacking the C-terminal PBC region (EGFP-ΔN-Ect2-ΔPBC). A schematic of the constructs is shown in . Upper panels: single central z-plane, lower panels: side views show orthogonal (x-z) projections of the z-stack along the line indicated in the corresponding upper panels. Scale bars: 10 µm (xy), 5 µm (z), n=50-56 cells from 3 independent experiments. B) Average pulse frequency of the Rho activity sensor signal (mCherry-Rhotekin-RBD) in cells expressing the indicated Ect2 variants. Red dots mark cells that generate fast high-amplitude pulses. n ≥ 51 cells from 3 independent experiments. using one-way ANOVA followed by Tukey’s post hoc test. Bars indicate mean ± SEM. C, E, G) Representative TIRF images of cells expressing the Rho activity sensor (right) and the indicated constructs (left), respectively. Frame rate: 3 frames/min, scale bar = 20 µm. D, F, H) Mean normalized intensity values of the Rho sensor signal in the corresponding cell regions (white boxes) in (C, E, F) . I) Proposed interplay between Ect2 and pulsatory Rho contraction signal network dynamics. Left: Pulsatory Rho contraction depends on a positive feedback loop in which Lbc-GEFs are recruited to the plasma membrane through binding to active Rho (right angled blue arrow). Curved red arrow points to enzymatic activation. Middle: In contrast, Ect2 is constitutively associated with the plasma membrane, independently of active Rho . Increased local concentration of Ect2 may result in a higher effective on-rate, thereby conferring a kinetic advantage that can outcompete Lbc-GEFs and thereby suppress Rho activity pulses. Curved red arrows illustrate enzymatic activation and allosteric activation. Right: Loss of the C-terminal PBC reduces Ect2 plasma membrane association, shifting its properties towards a Lbc-GEF-like phenotype, that is based on plasma membrane recruitment to active Rho, and that stimulates Rho activity pulses.

Article Snippet: Human U2OS osteosarcoma cells (HTB-96; ATCC) were maintained at 37 °C and 5 % CO 2 humidified atmosphere using standard cell culture techniques (DMEM + GlutaMAXTM medium, 10 % FBS, Life technologies; Gibco).

Techniques: Transfection, Control, Variant Assay, Construct, Activity Assay, Expressing, Clinical Proteomics, Membrane, Binding Assay, Activation Assay, Concentration Assay

Journal: bioRxiv

Article Title: Label-Free AI-Classification of Subcellular Organelles Based on Optical Photothermal Infrared Images

doi: 10.64898/2026.06.02.729616

Figure Lengend Snippet: U-2 OS cells were cultured on CaF 2 coverslips, fixed with 4% PFA, and stained with organelle-specific fluorescent markers. Fluorescence labeled IR datasets were then collected using OPTIR. These datasets were systematically analyzed to identify the minimal set of unique parameters necessary to distinguish organelles within the training set, which comprises multiple cells. The extracted parameters were used for two models, including a Random Forest classifier and a U-Net convolutional neural network. Model accuracy was assessed by evaluating the ability of the algorithms to classify organelles in independent test data not included in the training set. An example of the classification output shows the model accurately identifying Golgi (green) and nucleus (blue) in comparison to the fluorescence.

Article Snippet: U-2 OS cells (ATCC HTB-96) were cultured in DMEM containing L-Glutamine, 4.5 g/L glucose, and sodium pyruvate (Corning 10013CM), supplemented with penicillin-streptomycin (ThermoFisher Scientific 15140122) at 100 U/mL and 10% fetal bovine serum (GibcoTM 10438026).

Techniques: Cell Culture, Staining, Fluorescence, Labeling, Comparison

Journal: bioRxiv

Article Title: Label-Free AI-Classification of Subcellular Organelles Based on Optical Photothermal Infrared Images

doi: 10.64898/2026.06.02.729616

Figure Lengend Snippet: A . Representative fluorescence image of a HEK 293 cell with fluorescent labeling of the nucleus (blue), nucleolus (red), and mitochondria (green) (scale bar = 10 μm). The region of interest (ROI) is outlined in white. B . Classification of the ROI within HEK 293 cells using random forest (RF) and U-Net models trained on U-2 OS cells. The top panel shows a magnified fluorescent ROI, followed by random forest and U-Net classification results for the nucleus (left), mitochondria (middle), and nucleolus (right). C . Representative fluorescence image of stress granules (green) induced by oxidative stress within the cell (scale bar = 10 μm) with cellular region outlined in white. U-Net prediction images for each organelle are shown, giving poor predictions for all organelles except the nucleolus. D . Normalized OPTIR spectra (top) and second derivative (bottom) for stress granules and both the stressed and healthy cytoplasm and nucleus. Changes in protein secondary structure for beta sheet at 1643 cm -1 and alpha helices at 1656 cm -1 E . The ratio of the second derivative at 1643 cm -1 and 1656 cm -1 is compared. Stressed organelles show a decrease in this ratio compared to healthy spectra, which is paired with a large increase compared to the healthy cytoplasm.

Article Snippet: U-2 OS cells (ATCC HTB-96) were cultured in DMEM containing L-Glutamine, 4.5 g/L glucose, and sodium pyruvate (Corning 10013CM), supplemented with penicillin-streptomycin (ThermoFisher Scientific 15140122) at 100 U/mL and 10% fetal bovine serum (GibcoTM 10438026).

Techniques: Fluorescence, Labeling