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ATCC resource source identifier u2 os cells atcc rrid cvcl 0042 u2 os irhom1 irhom2 ko cells
Resource Source Identifier U2 Os Cells Atcc Rrid Cvcl 0042 U2 Os Irhom1 Irhom2 Ko Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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(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.

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

(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.

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.

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

(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.

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.

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

(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.

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.

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

(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.

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.

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

(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).

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).

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

(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.

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.

Techniques: Expressing, Mutagenesis, Fluorescence, Staining, Labeling

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