human osteosarcoma u2os cells  (ATCC)

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A) MTORC1 and AMPK activity is influenced by amino acids and glucose, respectively, and regulate ULK1 activity, which induces autophagy. (B-E) U2OS cells were treated with full media (FM), FM lacking glucose (-Glc), or FM lacking amino acids (-aa) for the indicated times and cell lysates analyzed by immunoblot for ACC, total and phosphorylated at S79 (B, D) , and S6K1 phosphorylated at T389 and actin as a loading control (C, E) . Immunoblots were quantified and signal normalized to total ACC (for D) or actin loading control (for E) plotted. Symbols represent mean of 3 independent experiments and bars are s.e.m. (F-I) Monoclonal U2OS cell lines expressing DFCP1 (F) , WIPI2B (G) , WIPI1 (H) , or ATG5 ( I ) were treated with FM (blue), -Glc (green), or -aa media (red) for 6 hours and subjected to live-cell fluorescent imaging. GFP-puncta (objects) for each reporter were quantified from single cells. Trajectories include mean objects per cell (symbols); bars represent 95% CI. (J-K) GFP-LC3B objects were quantified from cells treated with FM, -aa, or -Glc in the presence of BafA1 (to prevent lysosome degradation) or a vehicle control in 2 hour increments (J) . The number of GFP-LC3 puncta synthesized (solid symbols) and degraded (open symbols) from time 0 min was calculated and plotted. The dashed lines demarcate where individual datasets were collected and data stitched together. Trajectories include mean puncta per cell (symbols); bars represent standard deviation.

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Activity Assay, Western Blot, Control, Expressing, Imaging, Synthesized, Standard Deviation

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A-B) U2OS cells were treated for 6 hours with FM (blue), indicated as 100% aa (the concentration found in RPMI-1640), or 10% (green), 5% (orange), or 0% (red) of that amino acid concentration. Cells were lysed and ULK1 phosphorylated at S758 quantified (relative to actin loading control and normalized to time 0 controls) (A) . Bars represent means of 3 biological replicates. The data in (A) was fit to a sigmoidal dose-response curve (dashed line) to generate an EC 50 of 6% aa (B) . (C-D) Cells were treated with the medias described in A and imaged live from hours 4-6 in the presence of BafA1 (as in ). GFP-LC3 puncta were quantified from cells and sum intensity plotted (this is the sum of the intensity of all GFP-positive pixels, an output used to avoid potential issues with aggregated vesicles). Trajectories include mean objects per cell (symbols); bars represent s.e.m.; black lines represent simple linear regression (C) . The GFP-LC3 synthesis rates from the linear regression lines in (C) across amino acid concentrations were fit to a sigmoidal dose-response curve (dashed line) to generate an EC 50 of 7% aa (D) . (E) The rate of GFP-LC3 synthesis (derived from linear regression analysis, shown in (C) and the relative level of pULK1-S758 (from A) plotted to show a negative, linear association (dashed line, r 2 = 0.815).

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Concentration Assay, Control, Derivative Assay

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A) GFP-WIPI1 (green, left Y-axis) and GFP-WIPI2B (purple, right Y-axis) object counts over the 6 hour -aa treatment were overlaid. The gray region indicates the immediate starvation period (0-1 hours), and the yellow highlights the period of delayed autophagy under sustained starvation (3-6 hours). (B) EGFP-2xFYVE puncta (PI(3)P-positive cell membranes) were quantified from cells under FM (blue) or -aa (red) treatment. Note a lack of substantial puncta increase in the immediate (0-1 hour) period (gray shading). (C) The fraction of cells containing at least 1 GFP-WIPI2B puncta is plotted with time of -aa starvation. The dashed line represents 50% of the cell population. (D) Representative EGFP-2xFYVE puncta in U2OS cells treated with a VPS34 inhibitor (1 μM compound 31, lower panels) or vehicle control (upper panels). Blue = Hoechst nuclear stain; green = EGFP-2xFYVE; captured with a 60x oil objective. Scale bars in left panels are 20 μm and scale bars in right panels (insets) are 5 μm. (E) GFP-LC3 synthesis with BafA1 in the presence of compound 31 (1 μM) or vehicle control. BafA1 was added for 1 hour during either the first hour of -aa starvation (“0-1 hr” bars) or after 4 hours of -aa starvation (“4-5 hr” bars). Data shown represent GFP-LC3 puncta synthesis relative to vehicle control. Symbols represent mean and bars are s.e.m. **** = adjusted p < 0.0001, one-way ANOVA.

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Control, Staining

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A) Cells were cultured with or without amino acids for 6 hours prior to a restimulation phase of 60 min with FM (containing amino acids). (B-C) Representative images of GFP-DFCP1 (B) or GFP-WIPI2B (C) puncta in U2OS cells that were starved of amino acids for 6 hours and subject to aa-restimulation for 0 min (left), 10 min (middle) or 20 min (right). Insets show 2x magnification of indicated region to highlight disappearance of puncta. (D-G) DFCP1 (D) , WIPI2B (E) , WIPI1 (F) , and LC3B (G) quantified from cells during the restimulation period following -aa (red) or FM (blue) treatments. Symbols are mean GFP-positive puncta per cell and bars are s.e.m. Solid lines are non-linear regression models (one phase exponential decay). Gray shaded area emphasizes restoration to FM levels within 20 min of aa restimulation.

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Cell Culture

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A) Schematic representation of the time-of-drug-addition assay. U2OS cells were treated with (B) 2 µM JG98 or (C) 1 µM JG345 at the indicated time points (conditions 1-8) or with the DMSO control. Virus inoculum (CHIKV-LR at MOI 1) was present for 1.5 h, after which the inoculum was removed, and fresh medium was added with or without the Hsp70 inhibitors or DMSO control. At 9 hpi, the infectious virus particle production was assessed using the plaque assay. Data are presented as mean±SEM from at least three independent experiments. One-way ANOVA was used to evaluate statistical differences from the non-treated control and was presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Control, Virus, Plaque Assay

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A) U2OS cells were treated with increasing concentrations of the Hsp70 inhibitors or the equivalent volume of the DMSO control. The metabolic activity of the cells was assessed after 16 hours (h) of treatment using the MTS assay and was normalized to the DMSO control. A decrease of 15% in metabolically active cells was considered non-toxic (dotted line represents 85%). (B-F) U2OS cells were infected with CHIKV-LR OPY1 at multiplicity of infection (MOI) 1 in the presence of increasing concentrations of the (B) DMSO control (0.002% matches 0.1 µM JG-compound, 0.01% DMSO matches 0.5 µM JG-compound, etc.) or the Hsp70 inhibitors concentrations of (C) JG18, (D) JG40, (E) JG98, (F) JG345. Supernatants were collected at 9 hpi, and the number of infectious CHIKV particles was quantified using plaque assay on Vero-WHO cells. (B-F) Percentage inhibition was determined relative to the DMSO control, and IC50 (which corresponds to a 50% reduction in viral titer) per Hsp70 inhibitor was determined via non-linear regression. Data are presented as mean±SEM from three independent experiments. (B) Statistical differences were determined using One-way ANOVA and were presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Control, Activity Assay, MTS Assay, Metabolic Labelling, Infection, Plaque Assay, Inhibition

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: U2OS cells were infected with the (A) African S27 CHIKV strain or the (B) Caribbean 99659 strain at MOI 1 and simultaneously treated with 10 µM, 10 µM, 2 µM, or 1 µM of JG18, JG40, JG98, or JG345, respectively, or the DMSO control. Production of infectious virus particles was assessed at 9 hpi. (C) U2OS cells were infected with DENV A2 (16681) at MOI 0.5 and treated with 10 µM JG18 or JG40, or the vehicle control. 24 hpi, supernatants were collected and DENV progeny production was analyzed via plaque assay on BHK-21 cells. Data are presented as mean±SEM from three independent experiments, and statistical differences were determined via one-way ANOVA and presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Virus, Plaque Assay

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: U2OS cells were treated with 2 µM JG98, 1 µM JG345, or the DMSO control during CHIKV infection at MOI 10. Virus production was measured at 9 hpi using a plaque assay. Data are presented as mean±SEM from at least three independent experiments. One-way ANOVA was used to evaluate statistical differences from the DMSO control and was presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Control, Infection, Virus, Plaque Assay

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A) The total intracellular vRNA and (B) genomic vRNA copy number were assessed at 4, 6, and 8 hpi in U2OS cells infected with CHIKV at MOI 10 and treated with vehicle control DMSO or 2μM JG98. Intracellular vRNA copies were quantified by RT-qPCR using specific primers against (A) E1 and (B) nsP1. (C) The number of subgenomic RNA copies was determined by subtracting the genomic vRNA copies from the total vRNA copies. Data is presented as mean ± SEM from at least three independent experiments. Student T-test was used to evaluate statistical differences from the DMSO control per timepoint and were presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: Gating strategy to determine the percentage of cells positive for CHIKV E2 and capsid expression in U2OS cells treated with JG98, JG345, or the DMSO control. (A) Gating for cells and exclusion of doublets. (B and C) Gating to determine the percentage of cells positive for (B) capsid and (C) E2 based on mock-infected cells.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Expressing, Control, Infection

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A-D) U2OS cells were infected with CHIKV at MOI 10 and treated with JG98 (2μM), JG345 (1μM), or the DMSO control. At 9hpi, E2 and capsid expression were assessed using flow cytometry to determine the (A and C) percentage of positive cells and (B and D) mean fluorescent intensity (MFI; geometric mean). (E and F) Representative western blot of capsid, E2, or vinculin expression from protein lysates of U2OS cells infected with CHIKV at MOI 10 and treated for 9 h with Hsp70 inhibitor JG-98 (2μM), JG-345 (1μM), or the DMSO control. (G and H) Quantification of Western blots from three independent experiments. Protein levels are normalized to vinculin and are expressed as relative protein level to vehicle control DMSO. Data are presented as mean±SEM from at least three independent experiments. One-way ANOVA was used to evaluate statistical differences from the DMSO control and was presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Expressing, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: U2OS cells were infected with the (A-C, G-I) CHIKV-LR or the (D-F) CHIKV S27 strain for 9 h in the presence of (A-F) 2 µM JG98, 1 µM JG345, (G-I) 20 µM VER-155008, or the DMSO control. Supernatants were collected and the number of (A, D, and G) infectious particles and (B, E, and H) secreted genome equivalent copies (GECs) were measured using plaque assay and RT-qPCR, respectively. (C, F, and I) Specific infectivity is depicted as the ratio between produced infectious particles and GECs. Data are presented as mean± SEM from three independent experiments. One-way ANOVA was used to evaluate statistical differences from the DMSO control and was given when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Plaque Assay, Quantitative RT-PCR, Produced

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: Metabolic activity of the U2OS cells was assessed after 16 hours of treatment with increasing concentrations of the VER155008 or the equivalent volume of the DMSO control. The percentage of metabolically active cells relative to the DMSO control. A decrease of 15% in metabolically active cells was considered non-toxic (dotted line represents 85%). Data are presented as mean±SEM from three independent experiments.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Activity Assay, Control, Metabolic Labelling

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: Metabolic activity of (A) U2OS and (B) HFF-1 cells was assessed after 16 h of treatment with increasing concentrations of JG231 or the equivalent volume of the DMSO control. The percentage of metabolically active cells is presented relative to the DMSO control. A decrease of 15% in metabolically active cells was considered non-toxic (dotted line represents 85%). (C) U2OS cells and (D) HFF-1 cells were infected with CHIKV-LR for 9 h in the presence of 2 µM JG231 or the DMSO control. Supernatants were collected, and the number of infectious particles in the supernatant was determined using the plaque assay. (E and F) Mouse skin explants were infected with 10 6 PFU/mL of the CHIKV 899 strain during treatment with 2 µM JG231 or an equal volume of DMSO. (E) Infectious virus production (tissue culture infectious dose 50% (TCID 50 )) and (F) total virus production (GEC) at 2 dpi is displayed per mg tissue. The dotted line indicates the limit of quantification (LOQ). To evaluate statistical differences from the DMSO control, we used a Student T-test, and differences are presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Activity Assay, Control, Metabolic Labelling, Infection, Plaque Assay, Virus

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–talin interaction controls FB formation and FN fibrillogenesis. (A) Representative images of U2OS TNS3KO cells expressing mCh-TNS3-WT or -L702E spread overnight on FN-coated glass-bottom dishes, before being stained for α5 integrins (SNAKA51). (B) Left panel: Quantification of the mean number of α5 integrin-positive adhesions in A; n = 37 (WT) and 47 (L702E) cells. Right panel: Histograms and associated Gaussian curve fits for the frequency of the normalized distance (percentage of maximum) to the cell edge of each α5 integrin–positive adhesion; n = 3,335 (WT, 37 cells) and 1,860 (L702E, 47 cells) adhesions, respectively. (C) Images (background-subtracted) of cell-derived FN fibrils (labeled with IST9 antibody) produced by TNS3KO cells expressing GFP-TNS3-WT or GFP-TNS3-L702E, spread overnight on FN-coated glass. (D) Left panel: Quantification of FN fibrils in 40 × 40 μm square areas applied to each image; n = 56 (WT) and 54 (L702E) cells. Right panel: Fold change in FN-covered area (normalized to WT). (E) Images of U2OS TNS3KO cells expressing mCh-TNS3-WT or mCh-TNS3-L702E spread overnight in serum-free medium on VN-coated glass-bottom dishes, before being fixed and stained for the FA marker vinculin. (F) Quantification of the mean number of vinculin-positive adhesions in E; n = 74 (WT) and 51 (L702E) cells. (G) Images of TLNKO cells co-expressing GFP-TLN1 constructs (WT, R8m, R11m, or R8m+R11m) with mCh-TNS3-WT. Cells co-expressing GFP-TLN1-WT and mCh-TNS3-L702E (in the red box) were used as a negative control as the complete disruption of the talin–tensin3 interaction. (H) Left panel: Quantification of TNS3-positive adhesions in G. n = 29 (WT), 27 (R8m), 29 (R11m), 37 (R8m+R11m), and 35 (L702E) cells. Middle and right panels: Gaussian curve fits and means for the normalized distance (percentage of maximum) to the cell edge of each TNS3-positive adhesion in G; n = 3,528 (WT), 1,720 (R8m), 1,433 (R11m), 1,030 (R8m+R11m), and 790 (L702E) adhesions from 29, 27, 29, 37, and 35 cells, respectively. ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001 (B and D: Mann–Whitney test; F: unpaired t test; H left panel: Ordinary one-way ANOVA test with Tukey’s multiple comparisons test; H right panel: Kruskal–Wallis test with Dunn’s multiple comparisons test). All results are collected from three independent experiments. Scale bars: 10 μm (A and E), 20 μm (G), and 5 μm (C). Error bars are the SEM.

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, Staining, Derivative Assay, Labeling, Produced, Marker, Construct, Negative Control, Disruption, 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 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: Tensin3 regulates integrin activity through its interaction with talin and integrin. (A) Representative images of TLNKO cells co-expressing GFP-TLN1 with mCh-vector or mCh-TNS3, respectively. Active β1 integrins were stained with 9EG7 antibody. (B) Quantification of the fold change in 9EG7-positive adhesion counts in A. The fold change was calculated by normalizing the values for cells expressing mCh-TNS3 to the mean value for cells expressing mCh-vector. (C) Representative integrin activation (β1, 9EG7 antibody) profiles of TLNKO cells co-expressing GFP-TLN1 with mCh-vector (light gray) or mCh-TNS3 (dark gray) as measured by flow cytometry. (D) Integrin (β1) activation index (normalized to cells expressing GFP-TLN1 and mCh-vector) calculated from triplicate experiments of C. (E) Schematic of the tensin3 deletion and mutation constructs used, with the TBS motif (red) and the integrin-binding PTB domain indicated by orange dashed lines. All constructs are N-terminally labeled with GFP or mCherry. (F) Images of U2OS TNS3KO cells expressing the mCh-TNS3 construct shown in E, or a vector control. Cells were plated overnight on FN-coated glass-bottom dishes and stained for active β1 integrin (9EG7). (G) Quantification of β1 integrin–positive adhesions in F. n = 66 (vector), 70 (WT), 58 (L702E), 55 (ΔPTB), and 58 (L702E+ΔPTB) cells. (H) Integrin activation index (normalized to cells expressing GFP-TLN1 and mCh-TNS3-WT) calculated from triplicate flow cytometry experiments of . (I) Schematic of the tensin3 C-terminal deletion construct used. Note that this construct is N-terminally labeled with GFP. (J) U2OS TNS3KO cells expressing GFP-TNS3 constructs shown in E and I were cultured overnight before treatment with blebbistatin (50 μM) or an equivalent volume of DMSO (shown in ) for 60 min. (K) Quantification of β1 integrin–positive adhesion in J. n = 53 (WT), 43 (L702E), 66 (ΔPTB), and 53 (Cterm) cells. All data are collected from three independent experiments. Scale bars: 10 μm (A and J); 20 μm (F). Error bars are the SEM; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001 (B, D: unpaired t test; G and K: Kruskal–Wallis test with Dunn’s multiple comparisons test; H: ordinary one-way ANOVA with Turkey’s multiple comparisons).

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, Staining, Activation Assay, Flow Cytometry, Mutagenesis, Construct, Binding Assay, Labeling, Control, Cell Culture

Journal: Biomedical Optics Express

Article Title: Maximising imaging volumes of expanded tissues for inverted fluorescence microscopy

doi: 10.1364/BOE.579043

Figure Lengend Snippet: (a) Schematic of the imaging configuration using eye gel as the immersion medium, with (b) the corresponding experimental PSF showing a distorted, aberrated profile. (c) U2OS cells labeled for microtubules ( α -tubulin) imaged with eye gel immersion, showing degraded image quality. (d) Schematic of the configuration using two stacked FEP films, with (e) the corresponding PSF exhibiting near-ideal lateral and axial profiles. (f) U2OS cells labeled for microtubules ( α -tubulin) imaged with water immersion and two FEP films, showing high image quality.

Article Snippet: Human U2OS osteosarcoma cells (ATCC) and HeLa FlipIn cells (gift from Simon Bullock) were cultured in DMEM-Glutamax (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37∘C with 5% CO2.

Techniques: Imaging, Labeling