Journal: International Journal of Genomics

Article Title: Integrative Single‐Cell and Bulk Transcriptomics Identifies a Proliferative Malignant State and Supports a Functional Contribution of TROAP in Osteosarcoma

doi: 10.1155/ijog/1407947

Figure Lengend Snippet: Integrated single‐cell landscape of human osteosarcoma. (A) Harmony‐integrated UMAP of 39,063 quality‐controlled cells colored by global cluster. (B) The same embedding colored by patient, showing cross‐patient mixing after integration. (C) Reviewed major‐cell‐type annotations displayed on the integrated UMAP; labels mark lineage centroids. (D) Relative abundance of major cell types in each patient. Colors are consistent between panels C and D.

Article Snippet: HOS and U2OS human osteosarcoma cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, Virginia, United States; HOS, CRL‐1543; U2OS, HTB‐96).

Techniques: Single Cell

Journal: International Journal of Genomics

Article Title: Integrative Single‐Cell and Bulk Transcriptomics Identifies a Proliferative Malignant State and Supports a Functional Contribution of TROAP in Osteosarcoma

doi: 10.1155/ijog/1407947

Figure Lengend Snippet: TROAP depletion reduces clonogenic growth, overall wound closure, migration, and invasion in osteosarcoma cells. (A) Immunoblot analysis of TROAP in HOS and U2OS cells transfected with siNC, siTROAP‐1, or siTROAP‐2; beta‐actin is the loading control. (B) Colony‐formation images and colony counts. (C) Scratch‐wound images at 0 and 24 h and overall wound closure. Because the assay was not proliferation‐matched, it is not interpreted as direct migration evidence. (D) Transwell migration and invasion images and cell counts. Data are mean + /−standard deviation from three independent biological experiments. One‐way ANOVA with Dunnett′s test compared each siTROAP group with siNC; ∗∗∗ p < 0.001.

Article Snippet: HOS and U2OS human osteosarcoma cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, Virginia, United States; HOS, CRL‐1543; U2OS, HTB‐96).

Techniques: Migration, Western Blot, Analysis, Transfection, Control, Colony Assay, Standard Deviation

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