Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A–B ) Measurement of total Orai1 levels in the plasma membrane of STX11-depleted cells by flow-cytometry. U2OS cells stably expressing Orai1-BBS-YFP were transduced with Scr (black) or STX11 (red) shRNA, stimulated with 1 µM TG (broken lines) or control (solid lines) and incubated with alpha-bungarotoxin alexa fluor 647 (BTX-AF647). Total Orai1-YFP levels ( A ) and BTX-AF647 binding to surface Orai1 ( B ) were measured where binding to wildtype U2OS cells was used as the background fluorescence. N=3 ( C ) Representative quantitative PCR to assess the efficiency of knockdown in shRNA-treated Orai1-BBS-YFP expressing U2OS cells from ( A–B ). Total RNA extracted from cells treated with STX11 shRNA was subjected to qPCR analysis using Taqman probes for STX11. Data were normalized to RPL30 housekeeping gene. ( D–E ) Measurement of thapsigargin (TG) induced SOCE in U2OS cells treated with STX11 RNAi in ( A–B ). ( D ) Representative averaged traces of Fura-2 calcium imaging assay performed on cells in panels ( A–B ). ( E ) Bar plot showing relative mean % SOCE ± SE from three independent experiments in ( D ). Figure 2—figure supplement 2—source data 1. Folder containing the original flow cytometry files and gating strategy for . Figure 2—figure supplement 2—source data 2. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , U2OS cell line , ATCC , HTB-96 (RRID: CVCL_0042 ) , .

Techniques: Clinical Proteomics, Membrane, Flow Cytometry, Stable Transfection, Expressing, Transduction, shRNA, Control, Incubation, Binding Assay, Fluorescence, Real-time Polymerase Chain Reaction, Knockdown, Analysis, Imaging

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , U2OS cell line , ATCC , HTB-96 (RRID: CVCL_0042 ) , .

Techniques:

Journal: bioRxiv

Article Title: TDP-43 amyloid conversion in cells tracks the net charge of a polyampholyte motif, not condensate solidification

doi: 10.64898/2026.10.15.759291

Figure Lengend Snippet: (A) Schematic of the charged cluster architecture in TDP-43 (top) and representative confocal images of U2OS cells expressing NLS-deficient GFP-tagged TDP-43 WT and its variants (ΔPA1, ΔPA2, ΔPAM and ΔPAMΔLCD) 48 h after transfection (bottom). GFP, DAPI and merged images are shown. Scale bar: 20 μm. (B) Representative confocal images of U2OS cells expressing NLS-deficient GFP-tagged TDP-43 WT and its PAM mutants (K263E, PAM 3R-E, K263E/3R-E, K263A, PAM 3R-A, K263A/3R-A, 2E-K and ΔPAM) 48 h after transfection (right), with the corresponding PAM sequences at left and substituted residues in capital letters. Cells were counterstained with DAPI. Scale bar: 20 μm. (C) Representative confocal images of U2OS cells expressing NLS-deficient GFP-tagged TDP-43 WT, ΔPAM, PAM 3R-E and PAM 3R-A 48 h after transfection, fixed and stained with ProteoStat and Hoechst. Scale bar: 20 μm. (D–G) Individual FRAP recovery curves of cytoplasmic condensates in U2OS cells expressing NLS-deficient GFP-tagged TDP-43 WT (D), ΔPAM (E), PAM 3R-E (F) and PAM 3R-A (G), 48 h after transfection. Every curve recorded for that construct is plotted (n = 20 condensates each). (H) Mobile fraction of the condensates in (D–G), taken as the fitted plateau of a one-phase association fit and expressed as a percentage of the pre-bleach intensity. Box plots are Tukey plots. n = 20 condensates per construct. Brown-Forsythe and Welch ANOVA with Dunnett’s T3, each construct versus wild type. ****P < 0.0001. (I) Recovery half-time of the condensates in (H), from the rate constant of the same fits, in seconds. Box plots and n as in (H). Brown-Forsythe and Welch ANOVA with Dunnett’s T3, each construct versus wild type. ns, not significant; ****P < 0.0001. (J) Mobile fraction against recovery half-time (logarithmic ordinate) for the individual condensates in (H) and (I). Small symbols, individual condensates: WT (green), ΔPAM (pink), PAM 3R-E (blue) and PAM 3R-A (light green). Large symbols, construct medians, with error bars showing the 95% confidence interval of the median in both dimensions. Shaded areas are convex hulls drawn as a visual guide, not confidence regions.

Article Snippet: U2OS cells (ATCC, HTB-96, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM with high glucose; Cytiva, USA SH30023.01) supplemented with 10% fetal bovine serum (FBS; Cytiva, USA, SH30088.03) and 1X antibiotic-antimycotic solution (Anti-Anti; Gibco, USA, 15240-062) at 37°C in a humidified incubator with 5% CO2.

Techniques: Confocal, Expressing, Transfection, Staining, Construct

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: Cell Painting PLUS reveals phenotypic signatures for nephrotoxicity profiling in human RPTEC/TERT1

doi: 10.64898/2026.10.06.756929

Figure Lengend Snippet: a Pearson correlation scatter plots for compounds sharing the same MoA, comparing U2OS and RPTEC data. Pearson correlation was calculated between morphological profiles of compounds sharing the same MoA. Each dot represents the correlation score for an individual MoA, comparing the U2OS data (y-axis) with the RPTEC data (x-axis). Dotted lines mark the threshold of 0.75, indicating high correlation. Coloured labels indicate the MoAs specifically discussed in the main text. b Correlation between PIK3CA antagonists in RPTEC and U2OS cells. Symbols represent normalised values of single morphological features, corresponding channels, regions and modules are encoded by colour, shape and letter, respectively. r indicates Person correlation coefficient. c Morphological profile heatmap (B-scores and robust z-scores) for PIK3CA antagonists in RPTEC and U2OS. Modules, regions, and channels are colour-coded. A black-outlined box highlights lysosome-related features. (B-scores and z-scores are clipped to the range [-6, +6], values > 6 are set to 6 and values < −6 are set to −6 for display). d Morphological profile heatmap for TOP1/2 antagonists in RPTEC and U2OS. Modules, regions, and channels are colour-coded as indicated in panel c. The black-outlined box highlights DNA-related features. Lyso=lysosomes; Mito=mitochondria; ER=endoplasmic reticulum; Golgi=Golgi apparatus.

Article Snippet: U2OS and RPTEC/TERT1 (RPTEC) cells were cultured as previously described ( ): The U2OS (HTB-96) cells were obtained from ATCC and RPTEC cell line was a gift from Bob van de Water (Leiden University, The Netherlands) ( ).

Techniques:

Journal: bioRxiv

Article Title: Cell Painting PLUS reveals phenotypic signatures for nephrotoxicity profiling in human RPTEC/TERT1

doi: 10.64898/2026.10.06.756929

Figure Lengend Snippet: a U2OS CPP screening workflow and representative images of solvent control (DMSO) in U2OS cells (a’). n = 4. Scale bar = 20 µm. Lyso=lysosomes; Mito=mitochondria; ER=endoplasmic reticulum; Golgi=Golgi apparatus. b Quantitative cell morphology comparison between U2OS and RPTEC in solvent control (DMSO). Nuclear and cell areas (in µm 2 ) are shown. Dots represent the solvent control wells, pooled from all biological replicates. Asterisks indicate significant differences based on Wilcoxon rank-sum test with p ≤ 0.001. b’ Representative images of RPTEC and U2OS cells in solvent control, depicting DNA channel (top) and overlay of the CPP staining cycle 1 and 2 (bottom). Note that a broader imaging area is displayed compared to panel a’ in order to provide an overview of cell density, scale bar = 500 µm. c Probability density of the percentage of total cells relative to solvent control (PercentCells) and of activity (expressed as % active features) after exposure to reference chemicals. A feature is considered active with B-score (RPTEC) or robust z-score (U2OS) ≥ 3. The y-axis indicates the relative probability density. Colours represent the different cell lines. d Scatter plots of PercentCells versus percentage of active features across RPTEC and U2OS cells. Correlation coefficients (r) are displayed. e Comparison of cytotoxicity and activity of reference chemicals across RPTEC and U2OS cells. A compound is considered cytotoxic if it results in reduced PercentCells below 50%. A compound is considered active if at least 1% of features shows a robust z-score or B-score ≥ 3 (representing activities ≥3xMAD of the DMSO control).

Article Snippet: U2OS and RPTEC/TERT1 (RPTEC) cells were cultured as previously described ( ): The U2OS (HTB-96) cells were obtained from ATCC and RPTEC cell line was a gift from Bob van de Water (Leiden University, The Netherlands) ( ).

Techniques: Screening, Solvent, Control, Morphology, Comparison, Staining, Imaging, Activity Assay

Journal: bioRxiv

Article Title: Cell Painting PLUS reveals phenotypic signatures for nephrotoxicity profiling in human RPTEC/TERT1

doi: 10.64898/2026.10.06.756929

Figure Lengend Snippet: The plots were originally included in our companion manuscript and are reproduced here for completeness. a Percent replicating score for active compounds at non-toxic concentrations across four biological replicates, demonstrating screening robustness under active conditions. b Representative heatmap showing the mean signal intensity per well in RingRegion for the channel Alexa 647 (lysosome stain) and the percentage of total cell numbers normalised to DMSO control on one screening plate for U2OS CPP. Unnormalised raw values for the lysosome stain feature are displayed. c Histograms of nuclear area and DNA signal (HOECHST) intensity. For U2OS cells, nuclei sizes were filtered using a nuclear area range of 120-1000 µm 2 to exclude dead cells or cells with fragmented nuclei.

Article Snippet: U2OS and RPTEC/TERT1 (RPTEC) cells were cultured as previously described ( ): The U2OS (HTB-96) cells were obtained from ATCC and RPTEC cell line was a gift from Bob van de Water (Leiden University, The Netherlands) ( ).

Techniques: Screening, Staining, Control

Journal: bioRxiv

Article Title: Cell Painting PLUS reveals phenotypic signatures for nephrotoxicity profiling in human RPTEC/TERT1

doi: 10.64898/2026.10.06.756929

Figure Lengend Snippet: The data shown in this figure were initially published in and re-analysed here for the purpose of direct cell-line comparison. a Workflow of the Cell Painting PLUS (CPP) screen published in : RPTEC/TERT1, MCF7/vBos and U2OS cells were cultivated on multi-well plates and exposed to a set of 15 control compounds for 48 h. Plate layout and compound concentrations are described in detail in the original publication. Tested compounds are listed in panel c. The data were normalised as described in the present publication using the Jupyter notebook. b Cell and nuclear areas in µm 2 in solvent control (DMSO). Individual points represent median value per single well, showing all wells pooled from all replicates (4 independent biological replicates, each with 24 DMSO wells). Red lines and red numbers indicate median. Cell lines are colour-coded. c The percentage of total cells relative to solvent control condition (PercentCells) and percentage of active features after exposure to the 15 CPP performance control compounds at all tested concentrations. A feature is considered active with a robust z-score ≥ 3 (representing activities ≥ 3×MAD of the DMSO control). Single points indicate the median value for each tested condition from all replicates (4 independent biological replicates, each with 3 technical replicates). Compound names are encoded by letters, concentrations are encoded by dot size. Cell lines are colour-coded. “r” indicates Pearson correlation coefficient. d Activity of compounds for the different cell lines: Proportion of active compounds across all tested concentrations (level 1 = lowest tested concentration, level 7 = highest tested concentration). A compound is considered active when at least 1 % of all features have a robust z-score ≥ 3 (representing activities ≥ 3×MAD of the DMSO control).

Article Snippet: U2OS and RPTEC/TERT1 (RPTEC) cells were cultured as previously described ( ): The U2OS (HTB-96) cells were obtained from ATCC and RPTEC cell line was a gift from Bob van de Water (Leiden University, The Netherlands) ( ).

Techniques: Comparison, Cell Painting, Control, Solvent, Activity Assay, Concentration Assay