human bone osteosarcoma cells  (ATCC)

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