nih3t3 mouse fibroblast cells  (ATCC)

Journal: Nucleic Acids Research

Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

doi: 10.1093/nar/gkag168

Figure Lengend Snippet: CU-rich RNA promotes heterochromatin condensate organization during differentiation. ( A ) Nuclei of C2C12 myotubes (MT) treated with 1.5% 1,6-hexanediol (1,6-HD) or 1.5% 2,5-hexanediol (2,5-HD). Left: Representative images of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar, 5 μm. Middle: Time-course quantification of the number of heterochromatin foci per nucleus. Right: Boxplots show foci area (μm 2 , y -axis) at 5, 10, and 15 min posttreatment ( x -axis). n = 55 nuclei, three biological replicates. ( B ) Representative live-cell images of Hoechst 33342-stained MT nuclei before and after 1,6-HD treatment (0 and 15 min, respectively), taken from . Arrows indicate changes in heterochromatin foci intensity (pink, increased; blue, decreased), and the red arrow highlights an alteration in chromocenter integrity. ( C ) Number of heterochromatin foci per nucleus in MT following recovery from 1.5% 1,6-hexanediol (1,6-HD) treatment for 15 min, measured at indicated time points. n = 68 nuclei, three biological replicates. ( D ) Representative images of nuclei of myoblast (MB) and MT with or without 1,6-HD treatment (1.5%, 15 min). Right: Quantification of the number of heterochromatin foci per nucleus. n = 40 (MB), n = 60 (MT), three biological replicates. ( E ) Quantification of colocalization between indicated proteins and DAPI foci in MT with or without 1,6-HD treatment (1.5%, 15 min), by Pearson’s correlation coefficients. n = 60, three biological replicates. ( F ) Boxplot showing the distribution of Z -score of the interchromosomal interaction frequencies in MB and MT. ( G ) RNA FISH using ChRO1 and LacZ biotinylated probes. Biotin signal was detected by Fluorescein-conjugated Avidin DCS and amplified with biotinylated anti-Avidin and additional Fluorescein Avidin DCS. Right: Quantification of colocalization between biotin signal and DAPI foci. n = 50 nuclei. ( H ) Number of heterochromatin foci per nucleus of mouse fibroblast cells (NIH3T3) with or without doxycyline (Dox)-induced ChRO1a expression and/or 1,6-HD treatment (1.5%, 15 min). (EV; empty vector). n = 50, three biological replicates. ( I ) Number of heterochromatin foci per nucleus in MB with or without Dox-induced ChRO1a fragment (1–413, CUR) expression and/or 1,6-HD treatment (1.5%, 15 min). n = 75, three biological replicates. Statistical analyses and data presentation details are described in the “Materials and methods” section.

Article Snippet: C2C12 murine myoblast cells and NIH3T3 mouse fibroblast cells were obtained from the American-type culture collection and grown in a growth medium (GM) consisting of Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) fetal bovine serum at 37°C and 5% CO 2 .

Techniques: Staining, Avidin-Biotin Assay, Amplification, Expressing, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Talin contains tensin-binding sites in the R3, R4, R7R8, and R11 domain. (A) Schematic representation of talin with the aa numbers of each domain indicated. (B) Schematic representation of tensin3. Talin-binding site (TBS), aa 692–718. (C) Mitochondrial targeting talin1 full-length and deletion constructs. The indicated talin sequences were inserted between EGFP (GFP) and cBAK, with the aa number of talin regions indicated below. (D) Constructs shown in C were expressed with mCh-IDR in NIH3T3 cells. Black boxes indicate colocalization, and gray boxes indicate no association. (E) Western blotting of mitochondrial pulldown experiments. Constructs as used in D were expressed in HEK293T cells. Whole cell lysates (wcl) and purified mitochondria were immunoblotted. Note that the double band for mCh-IDR is due to known mCherry degradation. (F) Representation of additional talin1 constructs. (G–I) NIH3T3 cells expressing the constructs shown in C and F with mCh-IDR. (J) Summary table of the mitochondrial targeting assays. All results are collected from three independent experiments. Scale bars (D and G–I), 5 μm. aa, amino acid. Source data are available for this figure: .

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Binding Assay, Construct, Western Blot, Purification, Expressing

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Tensin3 binds multiple talin rod domains. (A–C) Western blotting of the mitochondrial pulldown experiments in HEK293T cells with the same constructs used in , showing that R3, R4, and R11 interact with mCh-IDR. (D) Representative images of NIH3T3 cells co-expressing mCh-TNS3-cBAK with GFP-vector, GFP-R1R2, GFP-R1R3, GFP-R4R6, GFP-R5R6, GFP-R7R8, GFP-R9R10, GFP-R11DD, or GFP-R12DD, respectively. Black boxes indicate colocalization, and gray boxes indicate no association. Note that TNS3-cBAK recruits R1R3, R4R6, R7R8, and R11DD to the mitochondria, but not GFP-vector, R1R2, R5R6, R9R10, or R12DD. (E) Summary table of the MTS experiments in D. (F–I) Representative images of NIH3T3 cells co-expressing GFP-R1R3-cBAK (F), GFP-R4R6-cBAK (G), GFP-R7R8-cBAK (H), or GFP-R11DD-cBAK (I) with mCh-IDR-WT or mCh-IDR-L702E, respectively. Note that L702E abolishes mCh-IDR interactions with all talin1 truncation constructs. (J) Images of cells co-expressing mCh-TNS3-cBAK (WT or L702E) with GFP-TLN1-E1770A, respectively. L702E abolishes TNS3 colocalization with TLN1-E1770A at the mitochondria. All experiments are performed three times. Scale bars, 5 μm. Source data are available for this figure: .

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Construct, Expressing, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Structure-based mutations in tensin3 disrupt its interaction with talin. (A) Crystal structure of the talin R11R12–tensin3 TBS (aa 692–718) complex. (i) TBS (orange) forms a six-helix bundle with R11 (cyan). (ii) TBS engages the α2-α5 face of R11. (B) R11-TBS interface is stabilized by multiple electrostatic and hydrophobic interactions. K2024, K2119, and K2133 (black), form electrostatic contacts with D696, S698, and D710 (red). (C) Poisson–Boltzmann electrostatic distribution map of the tensin3-binding surface of R11. Tensin3 peptide is shown in sticky representation with the hydrophobic residues labeled (red). (D) GFP-TLN1-cBAK was co-expressed with mCh-IDR wild-type (WT), deletion of TBS (ΔTBS), or those carrying the point mutation L702E, I706E, or L707E, respectively, in NIH3T3 cells. Groups of GFP-cBAK and mCh-IDR-ΔTBS were used as negative controls. (E) Summary table of D. (F) Mitochondrial pulldown experiment with the constructs used in D. (G) Quantification of F from triplicate experiments. Data are normalized to WT. Error bars are SEM; ** indicates P < 0.01 (ordinary one-way ANOVA with Dunnett’s multiple comparisons). (H) Representative images of NIH3T3 cells expressing mCh-TNS3-WT-cBAK or mCh-TNS3-L702E-cBAK and GFP-TLN1 (left panel) or GFP-TLN2 (right panel). mCh-cBAK was used as a negative control to recruit GFP-TLN1 and GFP-TLN2 to the mitochondria. Data are collected from three independent experiments. Scale bars (D and H), 5 μm. Source data are available for this figure: .

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Binding Assay, Labeling, Mutagenesis, Construct, Expressing, Negative Control

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Structural characterization of the multidomain talin–tensin3 interaction. (A) Representative images of NIH3T3 cells co-expressing GFP-R11DD-cBAK WT or carrying K2024E, K2031E, or both (K2024E+K2031E) with mCh-IDR. (B) Mitochondrial pulldown experiment using the same constructs as in A. (C) AlphaFold3 model of the talin R8–tensin3 TBS complex. TBS (orange) was predicted to engage the α2-α3 face of the R8 bundle (cyan). K1500 and R1510 (brown) are highlighted on the R11 domain. (D) NIH3T3 cells co-expressing GFP-R7R8-cBAK WT or carrying K1500E or R1510E with mCh-IDR. (E) Mitochondrial pulldown experiment using constructs as in D. (F) Cells co-expressing mCh-TNS3-WT-cBAK and GFP-TLN1 constructs (R8m, K1500E; R11m, K2024E+K2031E). mCh-TNS3-L702E-cBAK was used as a negative control. (G and H) Overlay 1 H- 15 N HSQC spectra of 15 N-labeled talin R3 (G) and talin R4 (H), at a concentration of 200 µM, in the absence (blue) and presence (red) of tensin3 TBS peptide at a molar ratio of 1:2. Magnified views in the right panels show the cross-peaks corresponding to the residues K869 and V871 of talin R3 (G) and the residues S927 and G969 of talin R4 (H), illustrating the progressive chemical shift changes at peptide molar ratios of 0 (blue), 0.25 (orange), 0.5 (green), 1.0 (coral), and 2.0 (red). The HSQC spectra of R3 and R4 were recorded at 700 and 800 MHz, respectively. (I and J) Mapping of the residue-specific CSD (related to ) on the AlphaFold3 models of talin R3 (I) and R4 (J) colored in cyan, respectively, in complex with tensin3 TBS (orange). Residues with significant CSDs are colored red, using a red-white linear gradient scale with red corresponding to the maximum CSD and white to the threshold. Residues with CSDs below the threshold are colored in green. Images are generated using PyMOL. (K–M) ITC profiles of the talin R3 (K), R4 (L), and R7R8 (M) interaction with tensin3 TBS, respectively. The upper panels (and left panel in M) show the raw heat flow data obtained during the titration of 600 µM of tensin3 TBS peptide into 40 µM talin R3 (K) or R7R8 (M), or the titration of 450 µM of tensin3 TBS peptide into 30 µM talin R4 (L), respectively, at a temperature of 25°C. The lower panels (and right panel in M) represent the integrated heat per injection plotted against the molar ratio. The data were fitted using a single-site binding model with dissociation constants (K d ) of 20.5 ± 1.80 µM for the R3-TBS interaction (K), 23.8 ± 2.94 µM for the R4-TBS interaction (L), and 15.8 ± 0.90 µM for the R7R8-TBS interaction. All results are collected from three independent experiments. Scale bars (A, D, and F), 5 μm. CSD, chemical shift difference. Source data are available for this figure: .

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Construct, Negative Control, Labeling, Concentration Assay, Residue, Generated, Titration, Injection, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Tensin3 regulates β1 integrin activity in the presence of talin. (A) Images of TLNKO cells expressing GFP-vector control, GFP-TLN1, or GFP-TNS3. F-actin was visualized by phalloidin staining. (B) Representative integrin activation (β1) profiles of TLNKO cells expressing GFP-vector, GFP-TLN1, or GFP-TNS3 as measured by flow cytometry analysis. Red profiles are from cells expressing the indicated constructs, and gray profiles are from the non-transfected cells in the same samples. (C) Integrin activation index (normalized to cells expressing GFP-vector) calculated from triplicate experiments of B. (D) Representative images of TLNKO cells co-expressing GFP-TLN1 with mCh-vector or mCh-TNS3 constructs (shown in ), respectively. Activated β1 integrin was visualized by staining with 9EG7 antibody. (E) Quantification of 9EG7-positive adhesions in D pooled from three independent experiments; n = 42 (vector), 38 (WT), 41 (L702E), 46 (ΔPTB), and 40 (L702E+ΔPTB) cells. (F) Representative integrin activation profiles of TLNKO cells co-expressing GFP-TLN1 with different mCh-TNS3 constructs. (G) Mean fluorescence intensity of F. Red values are from transfected cells, and gray values are from the nontransfected cells in the same samples. Note that the quantification of the integrin activation index pooled from three replicates is shown in . (H) Representative images of U2OS TNS3KO cells expressing GFP-TNS3 constructs (WT, L702E, ΔPTB, or L702E+ΔPTB). Cells were cultured on FN-coated glass overnight before being treated with DMSO or blebbistatin (50 μM, shown in ) for 60 min. Actin and β1 integrin were visualized by staining with phalloidin and 9EG7 antibody. Note that all four GFP-TNS3 constructs were localized to adhesions when cells were treated with DMSO. (I and J) NIH3T3 cells transfected with GFP-TNS3 constructs (same as those used in H) were treated with DMSO (I) or blebbistatin (50 μM, J) for 60 min before fixation and stained for actin. (K) Quantification of GFP-TNS3–positive adhesions in J. Note that GFP-TNS3-WT– and GFP-TNS3-ΔPTB–positive adhesions largely remain after blebbistatin treatment, whereas GFP-TNS3-L702E–positive and GFP-TNS3-Cterm–positive adhesions mostly disappear. n = 64 (WT), 49 (L702E), 62 (ΔPTB), and 71 (Cterm) cells. All error bars are the SEM. ** indicates P < 0.01, and **** indicates P < 0.0001 (C: ordinary one-way ANOVA with Turkey’s multiple comparisons, E and K: Kruskal–Wallis test with Dunn’s multiple comparisons test). Data are collected from three independent experiments. Scale bars in A, D, and H–J, 10 μm.

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Control, Staining, Activation Assay, Flow Cytometry, Construct, Transfection, Fluorescence, Cell Culture

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Talin is a retention signal for tensin3 that controls mechanosensitive tensin3 condensation and the client protein recruitment. (A) Representative images of NIH3T3 cells expressing GFP-TNS3 at a lower (0 μm) and a higher (0.6 μm) focal plane, with dashed boxes zoomed to the right. Note that TNS3 spheres are observed in proximity to the adhesion plane and in the cytoplasm. (B) Representative images of NIH3T3 cells expressing GFP-TNS3 (green) with immunostaining of endosomal marker EEA1, lysosomal marker LAMP1, or active β1 integrins (magenta). (C) Images of an NIH3T3 cell forming GFP-TNS3 condensates, labeled with fluorescently conjugated WGA (shown in blue). (D) Correlation between the relative protein level of mCh-TNS3-WT or mCh-TNS3-L702E and the cellular coverage (%) of TNS3 condensates in NIH3T3 cells, represented by linear regressions with 95% confidence intervals. n = 181 cells (WT) and 168 cells (L702E); nonparametric Spearman’s correlation r = 0.8773 (WT) and 0.9013 (L702E). (E) Representative background-subtracted images of a HFF cell expressing GFP-LIMD1 with staining for endogenous tensin3. The dashed box is zoomed in on the right, with a yellow arrow above two tensin3 condensates. (F) Line profile for the yellow arrow in E. (G) Background-subtracted images of HFF cells plated overnight on FN-coated 5 or 50 kPa PAA hydrogels. Endogenous tensin3, green; paxillin, magenta. (H) Quantification of the mean condensate number in G. n = 40 (5 kPa) and 41 (50 kPa) cells. (I) Representative images of NIH3T3 cells expressing GFP-TNS3-WT in green with exogenously co-expressed proteins in magenta. The black box indicates recruitment to the TNS3 condensates, and the gray box indicates no recruitment. (J and K) Images of NIH3T3 cells forming the TNS3 condensates in green with immunofluorescence staining for stress granule protein G3BP1 and TDP43 (J), and for tyrosine-phosphorylated proteins (K) with an antibody that probes phosphotyrosine (clone 4G10) in magenta. (L) Images of NIH3T3 cells forming the TNS3-L702E condensates in green with exogenously co-expressed proteins in magenta. Note that the summary table for I–L is shown in . **** indicates P < 0.0001 (D: ANCOVA; H: Mann–Whitney test). Scale bars are 10 µm (A–C, G, and I–L) or 5 µm (E). ANCOVA, analysis of covariance.

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Immunostaining, Marker, Labeling, Staining, Immunofluorescence, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Tensin3 undergoes LLPS in cells in a talin-regulated manner. (A) Schematic of human tensin3 with predicted disorder degree by IUPred . Residues with a predicted disorder tendency higher than 0.5 are considered disordered. (B) Schematic of the tensin3 IDR deletion (ΔIDR, deletion of aa 351–1,152) construct used. Note that this construct is N-terminally labeled with mCh. (C) Representative images of NIH3T3 cells expressing mCh-TNS3-WT or mCh-TNS3-ΔIDR. The dashed boxes are zoomed in on the right. Note that deletion of the IDR abolished the formation of tensin3 condensates. (D i and ii) Image (i) of NIH3T3 cells co-expressing GFP-LIMD1 (green) and mCh-TNS3 (magenta). The dashed box is zoomed in on the right (ii), with a line profile below for the yellow arrow line. (E) Correlation between the relative protein level of mCh-TNS3 and the cellular coverage (%) of TNS3 condensates in individual NIH3T3 cells, represented by a linear regression with 95% confidence intervals. n = 181 cells; R 2 = 0.6870; r = 0.8773 (nonparametric Spearman’s correlation). (F) Time-lapse images of a small GFP-TNS3 condensate (indicated by white arrows) derived from adhesion sites in NIH3T3 cells, related to . (G) Time-lapse images of GFP-TNS3 condensates (highlighted by dashed white circles) fusing in NIH3T3 cells, related to . (H) Images of NIH3T3 cells expressing mCh-TNS3-WT or mCh-TNS3-L702E. (I) Quantification of H pooled from triplicate experiments. Note that condensates that are larger than 0.1 μm 2 with a circularity between 0.7 and 1 were quantified. Left panel: The percentage of transfected cells with TNS3 condensate formation in each area (610 × 499 μm). The boxes represent the 25–75th percentiles with the median indicated; the whiskers indicate the range of values; n = 56 (WT) and 41 (L702E) areas. Middle panel: The mean number of TNS3 condensates in cells. Error bars are the SEM. Right panel: The mean radius of TNS3 condensates in each cell. n = 68 (WT) and 100 (L702E) cells. (J) Time-lapse images of FRAP experiment in NIH3T3 cells expressing GFP-TNS3-WT or GFP-TNS3-L702E. (K) Quantification of recovery halftime (left and middle panels) and mobile fraction (right panel) in J pooled from triplicate experiments. n = 27 (WT, 15 cells) and 26 (L702E, 17 cells) adhesions. * indicates P < 0.05, and **** indicates P < 0.0001 (I right and left panel: Unpaired t test; I middle panel and K: Welch’s t test). Scale bar: 10 µm (C, D, and H); 5 μm (F and G); 3 μm (J).

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Construct, Labeling, Expressing, Derivative Assay, Transfection

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Small GFP-TNS3 condensate derived from adhesion sites, related to . Time-lapse movie of GFP-TNS3 condensate formation at adhesion sites and release into the cytoplasm in a NIH3T3 cell. The green box indicates the zoomed area in . Time interval: 5 s; total time: 5 min 50 s. Scale bar: 10 µm.

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Dynamic fusion event of GFP-TNS3 condensates, related to . Movie of exogenously expressed tensin3 condensates fusing into larger spheres in a NIH3T3 cell. The green box indicates the zoomed area in . Time interval: 5 s; total time: 5 min 15 s. Scale bar: 10 µm.

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Tensin3 LLPS is controlled by talin activity in response to rigidity sensing. (A) Confocal images of NIH3T3 cells expressing mCh-TNS3-WT or mCh-TNS3-L702E plated overnight on FN-coated 1.5 or 28 kPa PDMS dishes. (B) Quantification of the mean TNS3 condensate number in A. n = 83 (WT/1.5 kPa), 80 (WT/28 kPa), 83 (L702E/1.5 kPa), and 89 (L702E/28 kPa) cells. Note that condensates that are larger than 0.1 μm 2 with a circularity between 0.7 and 1 were quantified. (C) Images of endogenous tensin3 or GFP-TNS3 (green) with endogenous paxillin (magenta) in HFF cells (top panel) and NIH3T3 cells (bottom panel) plated on FN-coated glass, respectively. The dashed boxes are zoomed to the right, with condensates indicated by yellow arrows. (D) Quantification of the circularity of overexpressed GFP-TNS3 condensates in NIH3T3 cells, endogenous tensin3 condensates in HFF cells, and tensin3 adhesions in HFF cells. The boxes represent the 25–75th percentiles with the median indicated; the whiskers indicate the range of values. n = 717 (overexpressed condensates, 17 cells), 192 (endogenous condensates, 29 cells), and 4,883 (tensin3 adhesions, 33 cells). (E) Representative images of HFF cells plated on FN-coated glass (left panel). The dashed boxes are zoomed to the right panel, with tensin3 condensates (green) indicated by white arrows and a line profile below for the yellow arrow line. The lipid membrane is labeled with WGA (blue); the active β1 integrin (magenta) is stained using 9EG7 antibody. Note that the tensin3 condensate is not colocalized with vesicle structures indicated by WGA and active β1 integrin. (F) Images (background-subtracted) of HFF cells plated overnight on FN-coated 1.5 or 28 kPa PDMS dishes. Endogenous tensin3 (green) and paxillin (magenta) were visualized by staining. The dashed boxes are zoomed in below, with condensates indicated by yellow arrows. (G) Quantification of the mean number of endogenous tensin3 condensates in F. n = 33 (1.5 kPa) and 40 (28 kPa) cells. (H) Mitochondrial pulldown experiments using HEK293T cells expressing GFP-TLN1-cBAK (WT or E1770A) and mCh-IDR. (I) Quantification of H pooled from three replicates. Data are normalized to WT. Error bars are the SEM. All data are collected from three independent experiments. * indicates P < 0.05, and **** indicates P <0.0001 (B and D: Kruskal–Wallis test with Dunn’s multiple comparisons; G: Mann–Whitney test; I: unpaired t test). Scale bar: 10 µm (A, C, and F) and 5 µm (E). Source data are available for this figure: .

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Expressing, Membrane, Labeling, Staining, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Tensin3 LLPS compartmentalizes adhesion and signaling proteins. (A and B) Representative images of NIH3T3 cells forming GFP-TNS3-WT (A) or GFP-TNS3-L702E (B) condensates (green) with immunostaining of various proteins (magenta). The dashed boxes are zoomed to the right. The black box indicates the recruitment to condensates, and the gray box indicates no recruitment. Scale bars are 10 µm. (C) Summary table of colocalization analysis of exogenously expressed (left panel, images are included in ) or endogenous proteins (right panel, images are shown in ; and ) with GFP-TNS3-WT or GFP-TNS3-L702E condensates in NIH3T3 cells.

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Immunostaining