experimental models u2 os ecacc 92022711 panc 1 ecacc  (ATCC)

Journal: Journal of Cellular and Molecular Medicine

Article Title: Gene Expression Alterations Associated With Resveratrol‐Induced Antiproliferative Effects and S‐Phase Cell Cycle Arrest in Osteosarcoma Cancer Cells

doi: 10.1111/jcmm.71111

Figure Lengend Snippet: Hyperspectral Imaging (HSI) of RSV and intracellular localisation in both OS cell lines. (A) Dark‐field optical imaging captures the signal of pure RSV, and (B) the associated spectral library shows the intensity of scattered light across wavelengths. (C) Dark‐field images and Spectral Angle Mapping (SAM) analysis in OS cell lines (SAOS‐2 and U2‐OS) show no RSV‐associated signals in control samples (completely black images), while treated cells display coloured pixels matching the RSV spectral signature, confirming intracellular localisation of the compound. Imaging was performed using a 60× oil immersion objective to resolve RSV distribution and spectral characteristics. (D) Quantification of RSV‐positive pixels reveals a statistically significant increase in both treated OS cell lines, compared to controls (*** p < 0.001), confirming cellular uptake of RSV.

Article Snippet: In vitro assays were conducted using OS cell lines SAOS‐2 and U2‐OS (ATCC, Cat. no. HTB‐85; Cat. no. HTB‐96) [ ], along with human bone marrow‐derived mesenchymal stem cells (hBMSCs) (Lonza Milan, Italy, PT‐2501). hBMSCs were characterised by flow cytometry analysis (FCA) for MSC surface markers, including positive markers (CD29, CD73 and CD90) and negative markers (CD14 and CD45) [ ].

Techniques: Imaging, Optical Imaging, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Gene Expression Alterations Associated With Resveratrol‐Induced Antiproliferative Effects and S‐Phase Cell Cycle Arrest in Osteosarcoma Cancer Cells

doi: 10.1111/jcmm.71111

Figure Lengend Snippet: Effects of RSV on the proliferation, viability and cell cycle distribution of OS cell lines and hBMSCs. (A) The MTT assay was used to assess the effect of resveratrol (RSV, 1–1000 μM) on OS cell lines (SAOS‐2 and U2‐OS) and healthy hBMSCs over 24, 48 and 72 h. In SAOS‐2 cells, RSV reduced proliferation in a dose‐dependent manner, compared to control ( p < 0.001), with significant decreases at 100 and 1000 μM after 48 h, compared to other treatments (** p < 0.01). In U2‐OS cells, RSV significantly reduced viability at all concentrations, with more differences observed after 24 h at higher concentrations (*** p < 0.0001). hBMSCs showed no significant changes, except for a notable increase at 1000 μM (*** p < 0.0001). (B) The Live/Dead assay confirmed the cytotoxic effect of RSV (100 μM) on OS cells after 48 h, using green Cyto‐dye for live cells and red propidium iodide for dead cells. (C) Fluorescence image quantification showed a significant reduction in live cells (*** p < 0.0001) and an increase in dead cells (** p < 0.001; * p < 0.01) in RSV‐treated OS cell lines compared to the control. (D) The effect of RSV on the cell cycle was analysed by BrdU/PI staining and flow cytometry after 48 h of treatment with 100 μM. The cytogram displays that RSV treatment resulted in a significant accumulation of OS cells in the S phase and a decrease in the G0/G1 and G2/M phases, compared to untreated controls. (E) Statistical analysis confirmed a significant increase in the S phase (*** p < 0.0001) and a significant reduction in the G0/G1 phase (* p < 0.001; ***p < 0.0001) in both OS cell lines.

Article Snippet: In vitro assays were conducted using OS cell lines SAOS‐2 and U2‐OS (ATCC, Cat. no. HTB‐85; Cat. no. HTB‐96) [ ], along with human bone marrow‐derived mesenchymal stem cells (hBMSCs) (Lonza Milan, Italy, PT‐2501). hBMSCs were characterised by flow cytometry analysis (FCA) for MSC surface markers, including positive markers (CD29, CD73 and CD90) and negative markers (CD14 and CD45) [ ].

Techniques: MTT Assay, Control, Live Dead Assay, Fluorescence, Staining, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Gene Expression Alterations Associated With Resveratrol‐Induced Antiproliferative Effects and S‐Phase Cell Cycle Arrest in Osteosarcoma Cancer Cells

doi: 10.1111/jcmm.71111

Figure Lengend Snippet: RSV induces apoptosis in OS cell lines. (A) Flow cytometry with Annexin V/PI staining was used to assess apoptosis in OS cells treated with 100 μM RSV for 48 h. The analysis identified early apoptotic (Annexin V+), late apoptotic (Annexin V+/PI+) and necrotic (PI+) cells, with Annexin V and PI intensities plotted on the X and Y axes, respectively. (B) Quantification showed that RSV significantly increased late apoptotic and necrotic cells in both OS cell lines (* p < 0.0001 for SAOS‐2 and ** p < 0.01 for U2‐OS), compared to control. A decrease in early apoptotic cells is observed in U2‐OS (** p < 0.01). (C) Gene expression profiling after RSV treatment revealed 18 differentially expressed apoptotic genes in SAOS‐2 (12 upregulated, 6 downregulated) and 21 in U2‐OS (10 upregulated, 11 downregulated), based on a Log 2 FC > 1 or < −1. (D) Gene Set Enrichment Analysis (GSEA) grouped these modulated genes into four categories: Positive regulators of apoptosis, negative regulators, caspases and death domain receptors, showing both up‐ and downregulated genes in each cell line. (E) A Venn diagram showed 11 apoptotic genes commonly modulated in both OS cell lines, with 7 unique to SAOS‐2 and 9 to U2‐OS, suggesting shared and cell‐specific mechanisms of RSV‐induced apoptosis. (F) Immunostaining for caspase‐3/7 revealed increased activation in both RSV‐treated OS cell lines after 48 h. (G) Fluorescence quantification using ImageJ confirmed a significant rise in activated caspase‐3/7 levels (** p < 0.001) in both RSV‐treated OS cells compared to controls.

Article Snippet: In vitro assays were conducted using OS cell lines SAOS‐2 and U2‐OS (ATCC, Cat. no. HTB‐85; Cat. no. HTB‐96) [ ], along with human bone marrow‐derived mesenchymal stem cells (hBMSCs) (Lonza Milan, Italy, PT‐2501). hBMSCs were characterised by flow cytometry analysis (FCA) for MSC surface markers, including positive markers (CD29, CD73 and CD90) and negative markers (CD14 and CD45) [ ].

Techniques: Flow Cytometry, Staining, Control, Gene Expression, Immunostaining, Activation Assay, Fluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Gene Expression Alterations Associated With Resveratrol‐Induced Antiproliferative Effects and S‐Phase Cell Cycle Arrest in Osteosarcoma Cancer Cells

doi: 10.1111/jcmm.71111

Figure Lengend Snippet: RSV inhibits cell migration and modulates ECM related gene expression in OS cells lines. (A) Bright‐field images from a wound healing assay show that control OS cells fully close the wound by 72 h (T3), whereas RSV‐treated cells (100 μM) exhibit no wound closure at any time point 0‐72 h (T0–T3), indicating that RSV strongly inhibits cell migration. (B) Quantitative analysis confirms significant wound closure in control cells over time compared to baseline T0 (0h) (° p < 0.0001), with additional increases at 48 h (T2) and 72 h (T3) compared to 24 h (T1) (* p < 0.001). Complete closure is observed at 72 h (T3) in control cells. (C) ECM‐related gene expression analysis using RT 2 Profiler PCR Array shows differential expression in RSV‐treated cells: 43 genes are modulated in SAOS‐2 (29 upregulated, 14 downregulated) and 26 in U2‐OS (11 upregulated, 15 downregulated). (D) Gene Set Enrichment Analysis (GSEA) categorizes these genes into five functional groups: Cell–cell adhesion, ECM‐cell adhesion, ECM constituents, ECM remodelling and basement membrane components. Up‐ and downregulated genes are identified for each group in both cell lines. (E) A Venn diagram reveals 20 ECM‐related genes commonly modulated in both SAOS‐2 and U2‐OS, indicating shared pathways influenced by RSV, particularly those involved in cell adhesion and ECM remodelling.

Article Snippet: In vitro assays were conducted using OS cell lines SAOS‐2 and U2‐OS (ATCC, Cat. no. HTB‐85; Cat. no. HTB‐96) [ ], along with human bone marrow‐derived mesenchymal stem cells (hBMSCs) (Lonza Milan, Italy, PT‐2501). hBMSCs were characterised by flow cytometry analysis (FCA) for MSC surface markers, including positive markers (CD29, CD73 and CD90) and negative markers (CD14 and CD45) [ ].

Techniques: Migration, Gene Expression, Wound Healing Assay, Control, Quantitative Proteomics, Functional Assay, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: Gene Expression Alterations Associated With Resveratrol‐Induced Antiproliferative Effects and S‐Phase Cell Cycle Arrest in Osteosarcoma Cancer Cells

doi: 10.1111/jcmm.71111

Figure Lengend Snippet: RSV modulates the Wnt/β‐Catenin signalling pathway and affects vimentin expression and β‐catenin localisation in OS cells lines. (A) Real‐time PCR analysis shows that RSV treatment (100 μM, 48 h) significantly downregulates key genes of the Wnt/β‐catenin pathway in SAOS‐2 and U2‐OS cells, including CTNNB1 , MMP7 , MMP9 and CD44 (* p < 0.001), all associated with ECM degradation, stemness and invasiveness. Conversely, CDH1 (epithelial marker) is upregulated in both lines (* p < 0.001 for SAOS‐2 and ** p < 0.01 for U2‐OS), suggesting a shift toward an epithelial phenotype. WNT1 and VIM are significantly downregulated (* p < 0.01 and * p < 0.05, respectively) in both cell lines, with c‐MYC reduced in SAOS‐2 (* p < 0.01). (B) Immunocytochemistry reveals a notable decrease in Vimentin protein levels in RSV‐treated cells. Vimentin, a mesenchymal marker, appears less expressed, with treated cells showing morphological changes including elongated filaments and enlarged cell body and nucleus. (C) β‐catenin immunostaining indicates that RSV prevents its nuclear translocation, with the protein mainly localized at cell junctions and in the cytoplasm in treated cells, while in controls β‐catenin is predominantly nuclear, confirming RSV‐mediated inhibition of Wnt/β‐catenin signalling.

Article Snippet: In vitro assays were conducted using OS cell lines SAOS‐2 and U2‐OS (ATCC, Cat. no. HTB‐85; Cat. no. HTB‐96) [ ], along with human bone marrow‐derived mesenchymal stem cells (hBMSCs) (Lonza Milan, Italy, PT‐2501). hBMSCs were characterised by flow cytometry analysis (FCA) for MSC surface markers, including positive markers (CD29, CD73 and CD90) and negative markers (CD14 and CD45) [ ].

Techniques: Expressing, Real-time Polymerase Chain Reaction, Marker, Immunocytochemistry, Immunostaining, Translocation Assay, Inhibition

Journal: The Journal of cell biology

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1083/jcb.202507147

Figure Lengend Snippet: (A) Representative western blot showing pelleted actin in U2-OS cells following ionomycin treatment (left) or histamine treatment (right) for the indicated times (sec). Tubulin also shown, with undetectable levels in the pellet. Standard, 40 ng of purified actin or tubulin. (B) Left: Time-course plots showing changes in cytoplasmic actin (GFP-F-tractin) in U2-OS cells and in pelleted actin following ionomycin addition. Fluorescence intensity and pelleted actin levels were each normalized to time 0 (F/F 0 ). GFP-F-tractin data represent n=33 cells, and actin pelleting data are from n=4 experiments; error bars indicate the standard error of the mean (SEM). Right: Same as left, but following histamine addition, with GFP-F-tractin data representing n=38 cells and actin pelleting data from n=3 experiments; error bars indicate SEM. (C) Time-lapse montage of an U2-OS cell transiently transfected with GFP-F-tractin, then stimulated with 4 μM ionomycin (left) or with 100 μM histamine (right) at time 0. Insets are magnified views of the boxed region. Scale bar: 10 μm in overview, 5 μm in inset; boxed region: 12 × 12 μm.

Article Snippet: Human osteosarcoma U2-OS cells (ATCC HTB-96) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-013-CV) supplemented with 10% newborn calf serum (R&D Systems; S11250).

Techniques: Western Blot, Purification, Fluorescence, Transfection

Journal: The Journal of cell biology

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1083/jcb.202507147

Figure Lengend Snippet: (A) Schematic diagram of the INF2 domains, highlighting the regions targeted by polyclonal antibodies, the disease-associated residue R218, as well as the 1-420, ΔNT, and FFC constructs used in this study. Domain boundaries (human INF2): N-terminus, 1-34; DID, 35-259; FH1, 421-520; FH2, 535-941; DAD, 967-995. (B) Schematic of the cell-free assay. U2-OS INF2 KO cells stably expressing GFP-INF2-CAAX are homogenized, followed by low speed centrifugation (600xg), after which the pellet (LSP) is used. Actin polymerization activity of the LSP is measured using pyrene actin assay, in the presence of actin monomer (2 μM, 5% pyrene-labeled), capping protein (50 nM) and profilin (6 μM). (C) U2-OS INF2 KO cells stably expressing GFP-INF2-CAAX or GFP were treated with vehicle or 100 μM histamine for 30 seconds, then fixed and stained with TRITC-phalloidin (to visualize actin filaments) and DAPI (to stain the nucleus). Scale bar: 10 μm in overview, 5 μm in inset; boxed region: 12 × 12 μm. (D) Western blots of total cell lysate and LSP from either U2-OS INF2 KO cells or GFP-INF2-CAAX stable cells. Equal protein (5 μg) was loaded in each lane and blot was probed with the indicated antibodies. (E, F) Representative pyrene-actin polymerization assays using LSP from GFP-INF2-CAAX stable cells (E) or from INF2 KO cells (F). Each reaction contains 10 μg LSP protein, 2 μM added actin monomer (5% pyrene-labeled), 50 nM capping protein, and 6 μM profilin, with or without the indicated components (167 nM anti-DID antibody, 167 nM anti-FH1FH2 antibody, 1 μM calcium with 250 nM CALM, as specified). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment.

Article Snippet: Human osteosarcoma U2-OS cells (ATCC HTB-96) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-013-CV) supplemented with 10% newborn calf serum (R&D Systems; S11250).

Techniques: Cell-Free Assay, Residue, Construct, Stable Transfection, Expressing, Centrifugation, Activity Assay, Pyrene Actin Assay, Labeling, Staining, Western Blot

Journal: The Journal of cell biology

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1083/jcb.202507147

Figure Lengend Snippet: (A) Time-lapse montage of U2-OS INF2 KO cells transiently expressing mApple-F-tractin along with either GFP-INF2 wild-type or GFP-INF2 R218Q, stimulated with 100 μM histamine at time 0. Insets show magnified views of the boxed regions. Scale bar: 10 μm in overview, 5 μm in inset; boxed region: 12 × 12 μm. (B) Time-course plot showing changes in cytoplasmic actin (mApple-F-tractin) following histamine addition. Y-axis values represent fluorescence intensity at each time point, normalized to the maximum fluorescence intensity observed (20 sec time point in INF2 WT). Error bars represent the standard error of the mean (SEM) of the indicated cell numbers. (C, D) Pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells transiently expressing either GFP-INF2-CAAX WT (C) or GFP-INF2-CAAX R218Q (D), with the indicated components (330 nM anti-DID antibody, 209 nM anti-FH1FH2 antibody, or 1 μM calcium with 100 nM CALM). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment.

Article Snippet: Human osteosarcoma U2-OS cells (ATCC HTB-96) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-013-CV) supplemented with 10% newborn calf serum (R&D Systems; S11250).

Techniques: Mutagenesis, Activity Assay, Expressing, Fluorescence, Labeling

Journal: The Journal of cell biology

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1083/jcb.202507147

Figure Lengend Snippet: (A) Western blots of total cell lysate and LSP from U2-OS GFP-INF2-CAAX stable cells transfected with either control siRNA or CAP1/2 siRNAs. Equal protein (5 μg) was loaded in each lane and blotted with the indicated antibodies. (B, C) Pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS GFP-INF2-CAAX cells transfected with either control siRNA (B) or CAP1/2 siRNAs (C), with the indicated components (330 nM anti-DID antibody, 209 nM anti-FH1FH2 antibody, 1 μM calcium with 100 nM CALM). Experiments were performed four times with similar results, and the data shown are a representative experiment. (D) Pyrene-actin polymerization assay performed as in (B) and (C), without siRNA but including 1 μM recombinant CAP2 protein where indicated. Experiments were performed twice with similar results. Data shown are a representative experiment. (E) Time-lapse montage of U2-OS cells transfected with either control siRNA or CAP1/2 siRNAs, further transfected with mApple-F-tractin, and stimulated with 100 μM histamine at time 0. Insets show magnified views of the boxed regions. Scale bar: 10 μm in overview, 5 μm in inset: 12 × 12 μm. (F) Time-course plot showing changes in cytoplasmic actin intensity (mApple-F-tractin) following histamine addition in U2-OS control KD cells and CAP1/2 KD cells. Y-axis values represent fluorescence intensity at each time point normalized to time 0 (F/F 0 ). Error bars represent the standard error of the mean (SEM), of the stated number of cells analyzed. (G, H) U2-OS cells transfected with either control siRNA or CAP1/2 siRNA were fixed and stained with TRITC-phalloidin without stimulation (G), or with 100 μM histamine treatment for 30 seconds (H). Insets show magnified views of the boxed regions, as indicated by the corresponding numbers. N =nucleus. Scale bar: 20 μm in overview, 5 μm in inset; boxed region: 20 × 20 μm. (I, J) Quantification of actin filament intensity in the perinuclear region without stimulation (H) or with 100 μM histamine treatment for 30 seconds (I), normalized to nuclear intensity. Each point represents one region of interest (ROI) per cell. P -value was calculated from an unpaired t -test. n.s. =not significant. error bars represent the standard error of the mean (SEM). Experiments were performed in triplicate.

Article Snippet: Human osteosarcoma U2-OS cells (ATCC HTB-96) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-013-CV) supplemented with 10% newborn calf serum (R&D Systems; S11250).

Techniques: Western Blot, Transfection, Control, Labeling, Polymerization Assay, Recombinant, Fluorescence, Staining

Journal: The Journal of cell biology

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1083/jcb.202507147

Figure Lengend Snippet: (A) Fluorescence anisotropy assay to measure actin monomer or INF2 DID binding by the INF2 C-terminal region or mDia1 DAD peptide. FITC- labeled INF2 C-terminal or TAMRA- labeled mDia1-DAD peptide (50 nM) were incubated with varying concentrations of LatA-stabilized actin or INF2 DID. (B) Pyrene-actin assays containing 2 μM actin monomers (5% pyrene labeled) and 2.5, 5, or 10 nM of the FFC construct of WT INF2 or the INF2-mDia1 DAD chimera. (C) Images from live-cell experiments in which U2-OS INF2 KO cells were transfected with mApple-F-tractin and either GFP-INF2 CAAX WT or GFP-INF2 CAAX-mDia1-DAD chimera. Cells stimulated with 100 μM histamine at time 0. Insets show magnified views of the boxed regions. Scale bar: 10 μm in overview, 5 μm in inset; boxed region: 12 × 12 μm. (D) Time-course plot showing changes in cytoplasmic actin intensity (mApple-F-tractin) following histamine addition in U2-OS INF2 KO cells expressing either GFP-INF2 CAAX-WT or GFP-INF2 CAAX-mDia1-DAD. Y-axis values represent fluorescence intensity at each time point normalized to time 0 (F/F 0 ). Error bars represent the standard error of the mean (SEM), of the stated number of cells analyzed. (E, F) U2-OS INF2 KO cells transfected with either GFP-INF2 CAAX-WT or GFP-INF2 CAAX-mDia1 DAD were fixed and stained with TRITC-phalloidin without stimulation (D), or with 100 μM histamine treatment for 30 seconds (E). Insets show magnified views of the boxed regions, as indicated by the corresponding numbers. N =nucleus. Scale bar: 10 μm in overview, 5 μm in inset; boxed region: 15 × 15 μm. (G, H) Quantification of actin filament intensity in the perinuclear region without stimulation (F) or with 100 μM histamine treatment for 30 seconds (G), normalized to nuclear intensity. Each point represents one region of interest (ROI) per cell. P -value was calculated from an unpaired t -test. n.s. =not significant. error bars represent the standard error of the mean (SEM). (I) Western blot of LSP from U2-OS INF2 KO cells transiently expressing either GFP-INF2-CAAX WT or GFP-INF2 CAAX-mDia1-DAD cells. Equal protein (5 μg) was loaded in each lane and blot was probed with the indicated antibodies. (J, K) LSP pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells transiently expressing GFP-INF2-CAAX wild-type (J) or GFP-INF2 CAAX-mDia1 DAD (K), with the indicated components (330 nM anti-DID antibody or 1 μM calcium with 100 nM CALM). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment.

Article Snippet: Human osteosarcoma U2-OS cells (ATCC HTB-96) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-013-CV) supplemented with 10% newborn calf serum (R&D Systems; S11250).

Techniques: Activation Assay, Fluorescence, Binding Assay, Labeling, Incubation, Construct, Transfection, Expressing, Staining, Western Blot

Journal: The Journal of cell biology

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1083/jcb.202507147

Figure Lengend Snippet: (A) Fluorescence anisotropy assay to measure CALM binding by the INF2 N-term peptide (1-29 aa). FITC- labeled INF2 N-term was incubated with varying concentrations of CALM. Two conditions were used: Condition 1 was 100 nM FITC-INF2-NT and 50 μM free calcium (blue); Condition 2 was 10 nM FITC-INF2-NT and 1 μM free calcium (red). The K d app were 4.5 and 3.6 μM, respectively. (B) Images from a time-lapse movie of U2-OS INF2 KO cells transiently expressing mApple-F-tractin along with either GFP-INF2-CAAX wild-type, WLL or ΔNT mutant. Cells were stimulated with 100 μM histamine at time 0. Insets show magnified views of the boxed regions. Scale bar: 10 μm in overview, 5 μm in inset; boxed region: 12 × 12 μm. (C, D) Time-course plot showing changes in cytoplasmic actin (mApple-F-tractin) following histamine addition in GFP-INF2-CAAX wild-type cells, WLL mutant (C) or ΔNT mutant (D). Y-axis values represent fluorescence intensity at each time point normalized to time 0 (F/F 0 ). Error bars represent the standard error of the mean (SEM) for indicated number of cells. (E-G) Cell-free pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells transiently expressing GFP-INF2-CAAX wild-type (E), WLL mutant (F) or ΔNT mutant (G), with the indicated components (330 nM anti-DID antibody, 209 nM anti-FH1FH2 antibody, or 1 μM calcium with 100 nM CALM). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment. (H, I) Cell-free pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells transiently expressing GFP-INF2-CAAX wild-type (H), or ΔNT mutant (I), with the indicated components (5 μM or 10 μM INF2 N-terminal peptide).

Article Snippet: Human osteosarcoma U2-OS cells (ATCC HTB-96) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-013-CV) supplemented with 10% newborn calf serum (R&D Systems; S11250).

Techniques: Activity Assay, Fluorescence, Binding Assay, Labeling, Incubation, Expressing, Mutagenesis

Journal: The Journal of cell biology

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1083/jcb.202507147

Figure Lengend Snippet: (A) Time-lapse montage of U2-OS INF2 KO cells transiently expressing mApple-F-tractin along with either GFP-INF2-CAAX wild-type, NM or LM mutant. Cells were stimulated with 100 μM histamine at time 0. Insets show magnified views of the boxed regions. Scale bar: 10 μm in overview, 5 μm in inset; boxed region: 12 × 12 μm. (B) Time-course plots showing changes in cytoplasmic actin filaments (mApple-F-tractin) following histamine addition in GFP-INF2-CAAX wild-type, NM or LM mutant expressing cells. Y-axis values represent fluorescence intensity at each time point, normalized to the maximum fluorescence intensity observed. Error bars represent the standard error of the mean (SEM) of the indicated cell numbers. (C) U2-OS INF2 KO cells transiently expressing GFP-INF2-CAAX wild-type, NM or LM mutant were fixed and stained with TRITC-phalloidin without stimulation. Insets show magnified views of the boxed regions. N =nucleus. Scale bar: 20 μm in overview, 5 μm in inset; boxed region: 20 × 20 μm. (D) Quantification of actin filament intensity in the perinuclear region, normalized to nuclear intensity. Each point represents one region of interest (ROI) per cell. P -value calculated from an unpaired t -test. Error bars represent the standard error of the mean (SEM). (E,F) Cell-free pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells transiently expressing either GFP-INF2-CAAX wild-type (E), or LM mutant (F), with the indicated components (330 nM anti-DID antibody, 209 nM anti-FH1FH2 antibody, or 1 μM calcium with 100 nM CALM). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment.

Article Snippet: Human osteosarcoma U2-OS cells (ATCC HTB-96) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-013-CV) supplemented with 10% newborn calf serum (R&D Systems; S11250).

Techniques: Expressing, Mutagenesis, Fluorescence, Staining, Labeling