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metastatic mda mb 231 breast adenocarcinoma cells  (ATCC)


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    ATCC metastatic mda mb 231 breast adenocarcinoma cells
    Vinculin depletion increases <t>bioenergetics</t> <t>of</t> <t>MDA-MB-231</t> cells. (A) Representative western blot of MDA-MB-231 cells expressing PercevalHR and pHRed probes transfected with siRNA to knock down vinculin (siVcl), including a scrambled control (siCtrl). (B) Quantification of the PercevalHR ratiometric signal for MDA cells expressing PercevalHR and pHRed probes treated with siRNA to knock down vinculin, including a scrambled control (siCtrl) and siVcl ( N = 3, n = 91–92 cells). (C) Representative ratiometric heatmaps of siCtrl and siVcl MDA cells expressing PercevalHR and pHRed. Scale bar, 100 µm. (D) Mean intensity of 2-NBDG of cells on 2D glass surfaces ( N = 3, n = 70–89 cells). (E) Representative images of 2-NBDG staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (F) Mean intensity of TMRM of cells on 2D glass surfaces ( N = 3, n = 58–84 cells). (G) Representative images of TMRM staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (H) Mean intensity of 2-NBDG for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91–103 cells). (I) Representative images of 2-NBDG staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (J) Mean intensity of TMRM for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91 cells). (K) Representative images of TMRM staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (L) Mean intensity of 2-NBDG in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 89–91 cells). (M) Mean intensity of TMRM in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 90–94 cells). (N) Mean intensity of 2-NBDG in MCF10A cells on 2D glass surfaces ( N = 3, n = 88–90 cells). (O) Mean intensity of TMRM in MCF10A cells on 2D glass surfaces ( N = 3, n = 90–96 cells). (P) Mean intensity of 2-NBDG for MDA ( N = 3, n = 93–98 cells) or Vcl KO ( N = 3, n = 79–86 cells) cells grown either on a tissue culture–treated surface (Adherent) or in a nonadherent plate (Floating), including representative phase-contrast images of MDA or Vcl KO cells seeded on a tissue culture surface or suspended in an ultra-low attachment plate. Scale bar, 50 µm. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. ns = not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. siVcl, siRNA targeting vinculin. Source data are available for this figure: .
    Metastatic Mda Mb 231 Breast Adenocarcinoma Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 27228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK"

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202504025

    Vinculin depletion increases bioenergetics of MDA-MB-231 cells. (A) Representative western blot of MDA-MB-231 cells expressing PercevalHR and pHRed probes transfected with siRNA to knock down vinculin (siVcl), including a scrambled control (siCtrl). (B) Quantification of the PercevalHR ratiometric signal for MDA cells expressing PercevalHR and pHRed probes treated with siRNA to knock down vinculin, including a scrambled control (siCtrl) and siVcl ( N = 3, n = 91–92 cells). (C) Representative ratiometric heatmaps of siCtrl and siVcl MDA cells expressing PercevalHR and pHRed. Scale bar, 100 µm. (D) Mean intensity of 2-NBDG of cells on 2D glass surfaces ( N = 3, n = 70–89 cells). (E) Representative images of 2-NBDG staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (F) Mean intensity of TMRM of cells on 2D glass surfaces ( N = 3, n = 58–84 cells). (G) Representative images of TMRM staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (H) Mean intensity of 2-NBDG for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91–103 cells). (I) Representative images of 2-NBDG staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (J) Mean intensity of TMRM for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91 cells). (K) Representative images of TMRM staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (L) Mean intensity of 2-NBDG in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 89–91 cells). (M) Mean intensity of TMRM in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 90–94 cells). (N) Mean intensity of 2-NBDG in MCF10A cells on 2D glass surfaces ( N = 3, n = 88–90 cells). (O) Mean intensity of TMRM in MCF10A cells on 2D glass surfaces ( N = 3, n = 90–96 cells). (P) Mean intensity of 2-NBDG for MDA ( N = 3, n = 93–98 cells) or Vcl KO ( N = 3, n = 79–86 cells) cells grown either on a tissue culture–treated surface (Adherent) or in a nonadherent plate (Floating), including representative phase-contrast images of MDA or Vcl KO cells seeded on a tissue culture surface or suspended in an ultra-low attachment plate. Scale bar, 50 µm. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. ns = not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. siVcl, siRNA targeting vinculin. Source data are available for this figure: .
    Figure Legend Snippet: Vinculin depletion increases bioenergetics of MDA-MB-231 cells. (A) Representative western blot of MDA-MB-231 cells expressing PercevalHR and pHRed probes transfected with siRNA to knock down vinculin (siVcl), including a scrambled control (siCtrl). (B) Quantification of the PercevalHR ratiometric signal for MDA cells expressing PercevalHR and pHRed probes treated with siRNA to knock down vinculin, including a scrambled control (siCtrl) and siVcl ( N = 3, n = 91–92 cells). (C) Representative ratiometric heatmaps of siCtrl and siVcl MDA cells expressing PercevalHR and pHRed. Scale bar, 100 µm. (D) Mean intensity of 2-NBDG of cells on 2D glass surfaces ( N = 3, n = 70–89 cells). (E) Representative images of 2-NBDG staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (F) Mean intensity of TMRM of cells on 2D glass surfaces ( N = 3, n = 58–84 cells). (G) Representative images of TMRM staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (H) Mean intensity of 2-NBDG for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91–103 cells). (I) Representative images of 2-NBDG staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (J) Mean intensity of TMRM for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91 cells). (K) Representative images of TMRM staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (L) Mean intensity of 2-NBDG in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 89–91 cells). (M) Mean intensity of TMRM in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 90–94 cells). (N) Mean intensity of 2-NBDG in MCF10A cells on 2D glass surfaces ( N = 3, n = 88–90 cells). (O) Mean intensity of TMRM in MCF10A cells on 2D glass surfaces ( N = 3, n = 90–96 cells). (P) Mean intensity of 2-NBDG for MDA ( N = 3, n = 93–98 cells) or Vcl KO ( N = 3, n = 79–86 cells) cells grown either on a tissue culture–treated surface (Adherent) or in a nonadherent plate (Floating), including representative phase-contrast images of MDA or Vcl KO cells seeded on a tissue culture surface or suspended in an ultra-low attachment plate. Scale bar, 50 µm. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. ns = not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. siVcl, siRNA targeting vinculin. Source data are available for this figure: .

    Techniques Used: Western Blot, Expressing, Transfection, Knockdown, Control, Staining, Whisker Assay

    Representative western blots. (A) Representative western blot confirming Vcl knockdown in MDA-MB-231 cells expressing PercevalHR and pHRed probes, including a scrambled control (siCtrl) and siVcl. Relevant samples are outlined with a red box, and uncropped membrane is included to show the ladder. (B and C) Representative western blot and (C) quantification from three independent western blots confirming Vcl KO and rescue in MDA-MB-231 cells ( N = 3). The Pre-Sort population refers to the Vcl KO cells transduced with Vcl-Venus before positively expressing Venus cells were sorted using FACS. Bar graph denotes the mean ± SEM. ns = not significant. (D) Representative western blot to confirm Vcl KO in NIH/3T3 cells. (E) Representative western blot to confirm Vcl KO in MCF10A cells. siVcl, siRNA targeting vinculin; FACS, fluorescence-activated cell sorting. Source data are available for this figure: .
    Figure Legend Snippet: Representative western blots. (A) Representative western blot confirming Vcl knockdown in MDA-MB-231 cells expressing PercevalHR and pHRed probes, including a scrambled control (siCtrl) and siVcl. Relevant samples are outlined with a red box, and uncropped membrane is included to show the ladder. (B and C) Representative western blot and (C) quantification from three independent western blots confirming Vcl KO and rescue in MDA-MB-231 cells ( N = 3). The Pre-Sort population refers to the Vcl KO cells transduced with Vcl-Venus before positively expressing Venus cells were sorted using FACS. Bar graph denotes the mean ± SEM. ns = not significant. (D) Representative western blot to confirm Vcl KO in NIH/3T3 cells. (E) Representative western blot to confirm Vcl KO in MCF10A cells. siVcl, siRNA targeting vinculin; FACS, fluorescence-activated cell sorting. Source data are available for this figure: .

    Techniques Used: Western Blot, Knockdown, Expressing, Control, Membrane, Transduction, Fluorescence, FACS

    Characterization of the MDA-MB-231 Vcl KO cell line. (A) Quantification of the percentage of dead cells from 30 fields of view per condition using a LIVE/DEAD kit ( N = 3). (B) Representative images of cell lines indicated treated with a LIVE/DEAD kit to assess cell viability in 2D. The right panel represents the positive control condition. Scale bar, 200 µm. (C and D) Percentage of EdU incorporation in parental MDA-MB-231 and Vcl KO cells and (D) representative images of EdU incorporation and DAPI to assess cell proliferation ( N = 3). Scale bar, 100 µm. Graphs denote the mean ± SEM. ns = not significant.
    Figure Legend Snippet: Characterization of the MDA-MB-231 Vcl KO cell line. (A) Quantification of the percentage of dead cells from 30 fields of view per condition using a LIVE/DEAD kit ( N = 3). (B) Representative images of cell lines indicated treated with a LIVE/DEAD kit to assess cell viability in 2D. The right panel represents the positive control condition. Scale bar, 200 µm. (C and D) Percentage of EdU incorporation in parental MDA-MB-231 and Vcl KO cells and (D) representative images of EdU incorporation and DAPI to assess cell proliferation ( N = 3). Scale bar, 100 µm. Graphs denote the mean ± SEM. ns = not significant.

    Techniques Used: Positive Control

    Vcl KO does not increase migration speed, but reduces persistence of MDA-MB-231 cells. (A) Representative phase-contrast images of cell lines indicated. Scale bar, 100 µm. (B) Quantification of cell aspect ratio and cell spread area on a 2D surface ( N = 3, n > 108 cells). (C and D) Speed and (D) persistence of MDA and Vcl KO cells migrating on 2D surfaces. (E) Representative trajectories of cells moving on 2D surfaces. (F) Representative color-coded time lapses of cells migrating on 2D surfaces where each cell outline represents a 50-min interval from the previous outline. Scale bar, 50 µm. (G) Magnitude of displacement from origin at each time point. Note: some SEM bars are too small to display ( N = 3, n = 78–95 cells for C–G). (H) Quantification of invasive fractions of MDA and Vcl KO cells through collagen-coated transwells ( N = 3, n = 6). (I and J) Speed and (J) persistence of cells migrating in a 1.5 mg/ml collagen matrix ( N = 3, n = 42–52 cells). (K) Representative XY trajectories of cells moving in a 1.5 mg/ml collagen matrix. (L) Cell aspect ratio and spread area in a 1.5 mg/ml collagen matrix ( N = 3, n = 42–52 cells). The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. XY plot shows the mean ± SEM. ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.
    Figure Legend Snippet: Vcl KO does not increase migration speed, but reduces persistence of MDA-MB-231 cells. (A) Representative phase-contrast images of cell lines indicated. Scale bar, 100 µm. (B) Quantification of cell aspect ratio and cell spread area on a 2D surface ( N = 3, n > 108 cells). (C and D) Speed and (D) persistence of MDA and Vcl KO cells migrating on 2D surfaces. (E) Representative trajectories of cells moving on 2D surfaces. (F) Representative color-coded time lapses of cells migrating on 2D surfaces where each cell outline represents a 50-min interval from the previous outline. Scale bar, 50 µm. (G) Magnitude of displacement from origin at each time point. Note: some SEM bars are too small to display ( N = 3, n = 78–95 cells for C–G). (H) Quantification of invasive fractions of MDA and Vcl KO cells through collagen-coated transwells ( N = 3, n = 6). (I and J) Speed and (J) persistence of cells migrating in a 1.5 mg/ml collagen matrix ( N = 3, n = 42–52 cells). (K) Representative XY trajectories of cells moving in a 1.5 mg/ml collagen matrix. (L) Cell aspect ratio and spread area in a 1.5 mg/ml collagen matrix ( N = 3, n = 42–52 cells). The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. XY plot shows the mean ± SEM. ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.

    Techniques Used: Migration, Whisker Assay

    Representative phase-contrast time lapse of an MDA-MB-231 cell migrating in 2D, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse depicts the MDA-MB-231 cell in . Scale bar, 50 µm. Playback, 7 fps.
    Figure Legend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 cell migrating in 2D, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse depicts the MDA-MB-231 cell in . Scale bar, 50 µm. Playback, 7 fps.

    Techniques Used: Inverted Microscopy

    Representative phase-contrast time lapse of an MDA-MB-231 Vcl KO cell migrating in 2D, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse depicts the MDA-MB-231 Vcl KO cell in . Scale bar, 50 µm. Playback, 7 fps.
    Figure Legend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 Vcl KO cell migrating in 2D, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse depicts the MDA-MB-231 Vcl KO cell in . Scale bar, 50 µm. Playback, 7 fps.

    Techniques Used: Inverted Microscopy

    Representative phase-contrast time lapse of an MDA-MB-231 cell moving in a 1.5 mg/ml collagen matrix, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse supports data presented in . Scale bar, 20 µm. Playback, 7 fps.
    Figure Legend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 cell moving in a 1.5 mg/ml collagen matrix, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse supports data presented in . Scale bar, 20 µm. Playback, 7 fps.

    Techniques Used: Inverted Microscopy

    Representative phase-contrast time lapse of an MDA-MB-231 Vcl KO cell moving in a 1.5 mg/ml collagen matrix, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse supports data presented in . Scale bar, 20 µm. Playback, 7 fps.
    Figure Legend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 Vcl KO cell moving in a 1.5 mg/ml collagen matrix, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse supports data presented in . Scale bar, 20 µm. Playback, 7 fps.

    Techniques Used: Inverted Microscopy

    Vcl KO increases GTP-RhoA–mediated ROCK activity and cell blebbing in MDA-MB-231 cells. (A) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces, where each outline represents a 4-s interval. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (B) Representative time lapse indicating that blebs are forming in the Vcl KO cells as shown by separation of the membrane from the actin cortex labeled using MemGlow and LifeAct, respectively. Scale bar, 10 µm in the first image. Scale bar, 5 µm in the zoomed-in time-lapse images. (C) Representative phase-contrast images of cells treated with 20 µm Y-27632 (Y27) or the vehicle control (deionized water). Scale bar, 100 µm. (D) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces treated with 20 µm Y-27632 or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. For D, each outline represents a 4-s interval. (E) Quantification of the percentage of blebbing cells ( N = 3, n = 28–31 fields of view) and the number of blebs per minute per cell ( N = 3, n = 36 cells) for cells treated with 20 µm Y-27632 or the vehicle control. (F) Quantification of the percentage of blebbing cells in cells treated with 25 µm Blebb or the vehicle control ( N = 3, n = 29 fields of view). (G) Normalized GTP-loaded RhoA measured using absorbance at 490 nm for cells treated with 20 µm Y-27632 or the vehicle control ( N = 4). Bar graphs denote the mean ± SEM. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
    Figure Legend Snippet: Vcl KO increases GTP-RhoA–mediated ROCK activity and cell blebbing in MDA-MB-231 cells. (A) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces, where each outline represents a 4-s interval. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (B) Representative time lapse indicating that blebs are forming in the Vcl KO cells as shown by separation of the membrane from the actin cortex labeled using MemGlow and LifeAct, respectively. Scale bar, 10 µm in the first image. Scale bar, 5 µm in the zoomed-in time-lapse images. (C) Representative phase-contrast images of cells treated with 20 µm Y-27632 (Y27) or the vehicle control (deionized water). Scale bar, 100 µm. (D) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces treated with 20 µm Y-27632 or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. For D, each outline represents a 4-s interval. (E) Quantification of the percentage of blebbing cells ( N = 3, n = 28–31 fields of view) and the number of blebs per minute per cell ( N = 3, n = 36 cells) for cells treated with 20 µm Y-27632 or the vehicle control. (F) Quantification of the percentage of blebbing cells in cells treated with 25 µm Blebb or the vehicle control ( N = 3, n = 29 fields of view). (G) Normalized GTP-loaded RhoA measured using absorbance at 490 nm for cells treated with 20 µm Y-27632 or the vehicle control ( N = 4). Bar graphs denote the mean ± SEM. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

    Techniques Used: Activity Assay, Membrane, Labeling, Control

    Representative time lapse of an MDA-MB-231 Vcl KO cell expressing LifeAct (red) and stained with 20 nM MemGlow (green), a membrane dye. The time lapse was acquired on a Zeiss LSM800 confocal microscope at 5-s intervals and represents the MDA-MB-231 Vcl KO cell pictured in . Scale bar, 10 µm. Playback, 3 fps.
    Figure Legend Snippet: Representative time lapse of an MDA-MB-231 Vcl KO cell expressing LifeAct (red) and stained with 20 nM MemGlow (green), a membrane dye. The time lapse was acquired on a Zeiss LSM800 confocal microscope at 5-s intervals and represents the MDA-MB-231 Vcl KO cell pictured in . Scale bar, 10 µm. Playback, 3 fps.

    Techniques Used: Expressing, Staining, Membrane, Microscopy

    Representative time lapse of an MDA-MB-231 cell expressing LifeAct (red) and stained with 20 nM MemGlow (green), a membrane dye. The time lapse was acquired on a Zeiss LSM800 confocal microscope at 5-s intervals and represents the MDA-MB-231 cell pictured in . Scale bar, 10 µm. Playback, 3 fps.
    Figure Legend Snippet: Representative time lapse of an MDA-MB-231 cell expressing LifeAct (red) and stained with 20 nM MemGlow (green), a membrane dye. The time lapse was acquired on a Zeiss LSM800 confocal microscope at 5-s intervals and represents the MDA-MB-231 cell pictured in . Scale bar, 10 µm. Playback, 3 fps.

    Techniques Used: Expressing, Staining, Membrane, Microscopy

    Representative phase-contrast time lapse of MDA-MB-231 cells moving in 2D, treated with vehicle control for Y-27632 (deionized water) for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.
    Figure Legend Snippet: Representative phase-contrast time lapse of MDA-MB-231 cells moving in 2D, treated with vehicle control for Y-27632 (deionized water) for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Techniques Used: Control, Inverted Microscopy

    Representative phase-contrast time lapse of MDA-MB-231 Vcl KO cells moving in 2D, treated with vehicle control for Y-27632 (deionized water) for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.
    Figure Legend Snippet: Representative phase-contrast time lapse of MDA-MB-231 Vcl KO cells moving in 2D, treated with vehicle control for Y-27632 (deionized water) for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Techniques Used: Control, Inverted Microscopy

    Representative phase-contrast time lapse of MDA-MB-231 cells moving in 2D, treated with 20 µM Y-27632 for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.
    Figure Legend Snippet: Representative phase-contrast time lapse of MDA-MB-231 cells moving in 2D, treated with 20 µM Y-27632 for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Techniques Used: Inverted Microscopy

    Representative phase-contrast time lapse of MDA-MB-231 Vcl KO cells moving in 2D, treated with 20 µM Y-27632 for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.
    Figure Legend Snippet: Representative phase-contrast time lapse of MDA-MB-231 Vcl KO cells moving in 2D, treated with 20 µM Y-27632 for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Techniques Used: Inverted Microscopy

    Metabolic inhibition of Vcl KO cells reduces cell blebbing, and Rho activation of parental MDA-MB-231 cells increases cell blebbing and bioenergetics. (A and B) Representative (A) phase-contrast images (scale bar, 100 µm) and (B) color-coded time lapses of Vcl KO cells treated with oligomycin, iodoacetate, or the vehicle control for each drug. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (C) Percentage of blebbing cells treated with oligomycin ( N = 3, n = 33–34 fields of view), iodoacetate ( N = 3, n = 31 fields of view), or the vehicle and the number of blebs per cell per minute for Vcl KO cells treated with oligomycin ( N = 3, n = 32–33 cells), iodoacetate ( N = 3, n = 32–34 cells), or the vehicle control. (D) Cell spread area of Vcl KO cells treated with oligomycin ( N = 3, n = 92–97 cells), iodoacetate ( N = 3, n = 101–137 cells), or the vehicle control, and the aspect ratio of Vcl KO cells treated with oligomycin ( N = 3, n = 90–91 cells), iodoacetate ( N = 3, n = 94–134 cells), or the vehicle control. (E and F) Representative (E) phase-contrast images (scale bar, 100 µm) and (F) color-coded time lapses of parental MDA cells treated with Rho activator or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (G) Percentage of blebbing cells ( N = 3, n = 36 fields of view) or number of blebs per cell per minute ( N = 3, n = 36 cells) for MDA cells treated with Rho activator or the vehicle control. (H) Mean intensity of 2-NBDG in parental MDA cells treated with Rho activator or the vehicle control ( N = 3, n = 90–97 cells). (I) Mean intensity of TMRM in parental MDA cells treated with Rho activator or the vehicle control ( N = 3, n = 61–62 cells). (J) Cell spread area ( N = 3, n = 106–140 cells) and aspect ratio ( N = 3, n = 106–140 cells) of parental MDA cells treated with the Rho activator compared with Vcl KO cells. (K) Cell spread area of parental MDA cells being treated with 20 µm oligomycin or the vehicle control over time after trypsinization ( N = 3, n = 74–89 cells). (L) Cell spread area of parental MDA cells being treated with 10 µm iodoacetate or the vehicle control over time after trypsinization ( N = 3, n = 72–87 cells). For K and L, time of 0 h indicates when cells are initially seeded on tissue culture surface and not yet adhered or spread. Bar graphs denote the mean ± SEM. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). XY plots show the mean ± SEM. ns = not significant, **P < 0.01, ****P < 0.0001.
    Figure Legend Snippet: Metabolic inhibition of Vcl KO cells reduces cell blebbing, and Rho activation of parental MDA-MB-231 cells increases cell blebbing and bioenergetics. (A and B) Representative (A) phase-contrast images (scale bar, 100 µm) and (B) color-coded time lapses of Vcl KO cells treated with oligomycin, iodoacetate, or the vehicle control for each drug. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (C) Percentage of blebbing cells treated with oligomycin ( N = 3, n = 33–34 fields of view), iodoacetate ( N = 3, n = 31 fields of view), or the vehicle and the number of blebs per cell per minute for Vcl KO cells treated with oligomycin ( N = 3, n = 32–33 cells), iodoacetate ( N = 3, n = 32–34 cells), or the vehicle control. (D) Cell spread area of Vcl KO cells treated with oligomycin ( N = 3, n = 92–97 cells), iodoacetate ( N = 3, n = 101–137 cells), or the vehicle control, and the aspect ratio of Vcl KO cells treated with oligomycin ( N = 3, n = 90–91 cells), iodoacetate ( N = 3, n = 94–134 cells), or the vehicle control. (E and F) Representative (E) phase-contrast images (scale bar, 100 µm) and (F) color-coded time lapses of parental MDA cells treated with Rho activator or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (G) Percentage of blebbing cells ( N = 3, n = 36 fields of view) or number of blebs per cell per minute ( N = 3, n = 36 cells) for MDA cells treated with Rho activator or the vehicle control. (H) Mean intensity of 2-NBDG in parental MDA cells treated with Rho activator or the vehicle control ( N = 3, n = 90–97 cells). (I) Mean intensity of TMRM in parental MDA cells treated with Rho activator or the vehicle control ( N = 3, n = 61–62 cells). (J) Cell spread area ( N = 3, n = 106–140 cells) and aspect ratio ( N = 3, n = 106–140 cells) of parental MDA cells treated with the Rho activator compared with Vcl KO cells. (K) Cell spread area of parental MDA cells being treated with 20 µm oligomycin or the vehicle control over time after trypsinization ( N = 3, n = 74–89 cells). (L) Cell spread area of parental MDA cells being treated with 10 µm iodoacetate or the vehicle control over time after trypsinization ( N = 3, n = 72–87 cells). For K and L, time of 0 h indicates when cells are initially seeded on tissue culture surface and not yet adhered or spread. Bar graphs denote the mean ± SEM. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). XY plots show the mean ± SEM. ns = not significant, **P < 0.01, ****P < 0.0001.

    Techniques Used: Inhibition, Activation Assay, Control, Whisker Assay



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    ATCC metastatic mda mb 231 cell line
    Vinculin depletion increases <t>bioenergetics</t> <t>of</t> <t>MDA-MB-231</t> cells. (A) Representative western blot of MDA-MB-231 cells expressing PercevalHR and pHRed probes transfected with siRNA to knock down vinculin (siVcl), including a scrambled control (siCtrl). (B) Quantification of the PercevalHR ratiometric signal for MDA cells expressing PercevalHR and pHRed probes treated with siRNA to knock down vinculin, including a scrambled control (siCtrl) and siVcl ( N = 3, n = 91–92 cells). (C) Representative ratiometric heatmaps of siCtrl and siVcl MDA cells expressing PercevalHR and pHRed. Scale bar, 100 µm. (D) Mean intensity of 2-NBDG of cells on 2D glass surfaces ( N = 3, n = 70–89 cells). (E) Representative images of 2-NBDG staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (F) Mean intensity of TMRM of cells on 2D glass surfaces ( N = 3, n = 58–84 cells). (G) Representative images of TMRM staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (H) Mean intensity of 2-NBDG for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91–103 cells). (I) Representative images of 2-NBDG staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (J) Mean intensity of TMRM for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91 cells). (K) Representative images of TMRM staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (L) Mean intensity of 2-NBDG in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 89–91 cells). (M) Mean intensity of TMRM in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 90–94 cells). (N) Mean intensity of 2-NBDG in MCF10A cells on 2D glass surfaces ( N = 3, n = 88–90 cells). (O) Mean intensity of TMRM in MCF10A cells on 2D glass surfaces ( N = 3, n = 90–96 cells). (P) Mean intensity of 2-NBDG for MDA ( N = 3, n = 93–98 cells) or Vcl KO ( N = 3, n = 79–86 cells) cells grown either on a tissue culture–treated surface (Adherent) or in a nonadherent plate (Floating), including representative phase-contrast images of MDA or Vcl KO cells seeded on a tissue culture surface or suspended in an ultra-low attachment plate. Scale bar, 50 µm. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. ns = not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. siVcl, siRNA targeting vinculin. Source data are available for this figure: .
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    Vinculin depletion increases bioenergetics of MDA-MB-231 cells. (A) Representative western blot of MDA-MB-231 cells expressing PercevalHR and pHRed probes transfected with siRNA to knock down vinculin (siVcl), including a scrambled control (siCtrl). (B) Quantification of the PercevalHR ratiometric signal for MDA cells expressing PercevalHR and pHRed probes treated with siRNA to knock down vinculin, including a scrambled control (siCtrl) and siVcl ( N = 3, n = 91–92 cells). (C) Representative ratiometric heatmaps of siCtrl and siVcl MDA cells expressing PercevalHR and pHRed. Scale bar, 100 µm. (D) Mean intensity of 2-NBDG of cells on 2D glass surfaces ( N = 3, n = 70–89 cells). (E) Representative images of 2-NBDG staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (F) Mean intensity of TMRM of cells on 2D glass surfaces ( N = 3, n = 58–84 cells). (G) Representative images of TMRM staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (H) Mean intensity of 2-NBDG for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91–103 cells). (I) Representative images of 2-NBDG staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (J) Mean intensity of TMRM for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91 cells). (K) Representative images of TMRM staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (L) Mean intensity of 2-NBDG in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 89–91 cells). (M) Mean intensity of TMRM in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 90–94 cells). (N) Mean intensity of 2-NBDG in MCF10A cells on 2D glass surfaces ( N = 3, n = 88–90 cells). (O) Mean intensity of TMRM in MCF10A cells on 2D glass surfaces ( N = 3, n = 90–96 cells). (P) Mean intensity of 2-NBDG for MDA ( N = 3, n = 93–98 cells) or Vcl KO ( N = 3, n = 79–86 cells) cells grown either on a tissue culture–treated surface (Adherent) or in a nonadherent plate (Floating), including representative phase-contrast images of MDA or Vcl KO cells seeded on a tissue culture surface or suspended in an ultra-low attachment plate. Scale bar, 50 µm. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. ns = not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. siVcl, siRNA targeting vinculin. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Vinculin depletion increases bioenergetics of MDA-MB-231 cells. (A) Representative western blot of MDA-MB-231 cells expressing PercevalHR and pHRed probes transfected with siRNA to knock down vinculin (siVcl), including a scrambled control (siCtrl). (B) Quantification of the PercevalHR ratiometric signal for MDA cells expressing PercevalHR and pHRed probes treated with siRNA to knock down vinculin, including a scrambled control (siCtrl) and siVcl ( N = 3, n = 91–92 cells). (C) Representative ratiometric heatmaps of siCtrl and siVcl MDA cells expressing PercevalHR and pHRed. Scale bar, 100 µm. (D) Mean intensity of 2-NBDG of cells on 2D glass surfaces ( N = 3, n = 70–89 cells). (E) Representative images of 2-NBDG staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (F) Mean intensity of TMRM of cells on 2D glass surfaces ( N = 3, n = 58–84 cells). (G) Representative images of TMRM staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (H) Mean intensity of 2-NBDG for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91–103 cells). (I) Representative images of 2-NBDG staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (J) Mean intensity of TMRM for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91 cells). (K) Representative images of TMRM staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (L) Mean intensity of 2-NBDG in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 89–91 cells). (M) Mean intensity of TMRM in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 90–94 cells). (N) Mean intensity of 2-NBDG in MCF10A cells on 2D glass surfaces ( N = 3, n = 88–90 cells). (O) Mean intensity of TMRM in MCF10A cells on 2D glass surfaces ( N = 3, n = 90–96 cells). (P) Mean intensity of 2-NBDG for MDA ( N = 3, n = 93–98 cells) or Vcl KO ( N = 3, n = 79–86 cells) cells grown either on a tissue culture–treated surface (Adherent) or in a nonadherent plate (Floating), including representative phase-contrast images of MDA or Vcl KO cells seeded on a tissue culture surface or suspended in an ultra-low attachment plate. Scale bar, 50 µm. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. ns = not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. siVcl, siRNA targeting vinculin. Source data are available for this figure: .

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Western Blot, Expressing, Transfection, Knockdown, Control, Staining, Whisker Assay

    Representative western blots. (A) Representative western blot confirming Vcl knockdown in MDA-MB-231 cells expressing PercevalHR and pHRed probes, including a scrambled control (siCtrl) and siVcl. Relevant samples are outlined with a red box, and uncropped membrane is included to show the ladder. (B and C) Representative western blot and (C) quantification from three independent western blots confirming Vcl KO and rescue in MDA-MB-231 cells ( N = 3). The Pre-Sort population refers to the Vcl KO cells transduced with Vcl-Venus before positively expressing Venus cells were sorted using FACS. Bar graph denotes the mean ± SEM. ns = not significant. (D) Representative western blot to confirm Vcl KO in NIH/3T3 cells. (E) Representative western blot to confirm Vcl KO in MCF10A cells. siVcl, siRNA targeting vinculin; FACS, fluorescence-activated cell sorting. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative western blots. (A) Representative western blot confirming Vcl knockdown in MDA-MB-231 cells expressing PercevalHR and pHRed probes, including a scrambled control (siCtrl) and siVcl. Relevant samples are outlined with a red box, and uncropped membrane is included to show the ladder. (B and C) Representative western blot and (C) quantification from three independent western blots confirming Vcl KO and rescue in MDA-MB-231 cells ( N = 3). The Pre-Sort population refers to the Vcl KO cells transduced with Vcl-Venus before positively expressing Venus cells were sorted using FACS. Bar graph denotes the mean ± SEM. ns = not significant. (D) Representative western blot to confirm Vcl KO in NIH/3T3 cells. (E) Representative western blot to confirm Vcl KO in MCF10A cells. siVcl, siRNA targeting vinculin; FACS, fluorescence-activated cell sorting. Source data are available for this figure: .

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Western Blot, Knockdown, Expressing, Control, Membrane, Transduction, Fluorescence, FACS

    Characterization of the MDA-MB-231 Vcl KO cell line. (A) Quantification of the percentage of dead cells from 30 fields of view per condition using a LIVE/DEAD kit ( N = 3). (B) Representative images of cell lines indicated treated with a LIVE/DEAD kit to assess cell viability in 2D. The right panel represents the positive control condition. Scale bar, 200 µm. (C and D) Percentage of EdU incorporation in parental MDA-MB-231 and Vcl KO cells and (D) representative images of EdU incorporation and DAPI to assess cell proliferation ( N = 3). Scale bar, 100 µm. Graphs denote the mean ± SEM. ns = not significant.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Characterization of the MDA-MB-231 Vcl KO cell line. (A) Quantification of the percentage of dead cells from 30 fields of view per condition using a LIVE/DEAD kit ( N = 3). (B) Representative images of cell lines indicated treated with a LIVE/DEAD kit to assess cell viability in 2D. The right panel represents the positive control condition. Scale bar, 200 µm. (C and D) Percentage of EdU incorporation in parental MDA-MB-231 and Vcl KO cells and (D) representative images of EdU incorporation and DAPI to assess cell proliferation ( N = 3). Scale bar, 100 µm. Graphs denote the mean ± SEM. ns = not significant.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Positive Control

    Vcl KO does not increase migration speed, but reduces persistence of MDA-MB-231 cells. (A) Representative phase-contrast images of cell lines indicated. Scale bar, 100 µm. (B) Quantification of cell aspect ratio and cell spread area on a 2D surface ( N = 3, n > 108 cells). (C and D) Speed and (D) persistence of MDA and Vcl KO cells migrating on 2D surfaces. (E) Representative trajectories of cells moving on 2D surfaces. (F) Representative color-coded time lapses of cells migrating on 2D surfaces where each cell outline represents a 50-min interval from the previous outline. Scale bar, 50 µm. (G) Magnitude of displacement from origin at each time point. Note: some SEM bars are too small to display ( N = 3, n = 78–95 cells for C–G). (H) Quantification of invasive fractions of MDA and Vcl KO cells through collagen-coated transwells ( N = 3, n = 6). (I and J) Speed and (J) persistence of cells migrating in a 1.5 mg/ml collagen matrix ( N = 3, n = 42–52 cells). (K) Representative XY trajectories of cells moving in a 1.5 mg/ml collagen matrix. (L) Cell aspect ratio and spread area in a 1.5 mg/ml collagen matrix ( N = 3, n = 42–52 cells). The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. XY plot shows the mean ± SEM. ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Vcl KO does not increase migration speed, but reduces persistence of MDA-MB-231 cells. (A) Representative phase-contrast images of cell lines indicated. Scale bar, 100 µm. (B) Quantification of cell aspect ratio and cell spread area on a 2D surface ( N = 3, n > 108 cells). (C and D) Speed and (D) persistence of MDA and Vcl KO cells migrating on 2D surfaces. (E) Representative trajectories of cells moving on 2D surfaces. (F) Representative color-coded time lapses of cells migrating on 2D surfaces where each cell outline represents a 50-min interval from the previous outline. Scale bar, 50 µm. (G) Magnitude of displacement from origin at each time point. Note: some SEM bars are too small to display ( N = 3, n = 78–95 cells for C–G). (H) Quantification of invasive fractions of MDA and Vcl KO cells through collagen-coated transwells ( N = 3, n = 6). (I and J) Speed and (J) persistence of cells migrating in a 1.5 mg/ml collagen matrix ( N = 3, n = 42–52 cells). (K) Representative XY trajectories of cells moving in a 1.5 mg/ml collagen matrix. (L) Cell aspect ratio and spread area in a 1.5 mg/ml collagen matrix ( N = 3, n = 42–52 cells). The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. XY plot shows the mean ± SEM. ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Migration, Whisker Assay

    Representative phase-contrast time lapse of an MDA-MB-231 cell migrating in 2D, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse depicts the MDA-MB-231 cell in . Scale bar, 50 µm. Playback, 7 fps.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 cell migrating in 2D, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse depicts the MDA-MB-231 cell in . Scale bar, 50 µm. Playback, 7 fps.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Inverted Microscopy

    Representative phase-contrast time lapse of an MDA-MB-231 Vcl KO cell migrating in 2D, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse depicts the MDA-MB-231 Vcl KO cell in . Scale bar, 50 µm. Playback, 7 fps.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 Vcl KO cell migrating in 2D, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse depicts the MDA-MB-231 Vcl KO cell in . Scale bar, 50 µm. Playback, 7 fps.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Inverted Microscopy

    Representative phase-contrast time lapse of an MDA-MB-231 cell moving in a 1.5 mg/ml collagen matrix, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse supports data presented in . Scale bar, 20 µm. Playback, 7 fps.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 cell moving in a 1.5 mg/ml collagen matrix, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse supports data presented in . Scale bar, 20 µm. Playback, 7 fps.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Inverted Microscopy

    Representative phase-contrast time lapse of an MDA-MB-231 Vcl KO cell moving in a 1.5 mg/ml collagen matrix, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse supports data presented in . Scale bar, 20 µm. Playback, 7 fps.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 Vcl KO cell moving in a 1.5 mg/ml collagen matrix, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse supports data presented in . Scale bar, 20 µm. Playback, 7 fps.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Inverted Microscopy

    Vcl KO increases GTP-RhoA–mediated ROCK activity and cell blebbing in MDA-MB-231 cells. (A) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces, where each outline represents a 4-s interval. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (B) Representative time lapse indicating that blebs are forming in the Vcl KO cells as shown by separation of the membrane from the actin cortex labeled using MemGlow and LifeAct, respectively. Scale bar, 10 µm in the first image. Scale bar, 5 µm in the zoomed-in time-lapse images. (C) Representative phase-contrast images of cells treated with 20 µm Y-27632 (Y27) or the vehicle control (deionized water). Scale bar, 100 µm. (D) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces treated with 20 µm Y-27632 or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. For D, each outline represents a 4-s interval. (E) Quantification of the percentage of blebbing cells ( N = 3, n = 28–31 fields of view) and the number of blebs per minute per cell ( N = 3, n = 36 cells) for cells treated with 20 µm Y-27632 or the vehicle control. (F) Quantification of the percentage of blebbing cells in cells treated with 25 µm Blebb or the vehicle control ( N = 3, n = 29 fields of view). (G) Normalized GTP-loaded RhoA measured using absorbance at 490 nm for cells treated with 20 µm Y-27632 or the vehicle control ( N = 4). Bar graphs denote the mean ± SEM. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Vcl KO increases GTP-RhoA–mediated ROCK activity and cell blebbing in MDA-MB-231 cells. (A) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces, where each outline represents a 4-s interval. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (B) Representative time lapse indicating that blebs are forming in the Vcl KO cells as shown by separation of the membrane from the actin cortex labeled using MemGlow and LifeAct, respectively. Scale bar, 10 µm in the first image. Scale bar, 5 µm in the zoomed-in time-lapse images. (C) Representative phase-contrast images of cells treated with 20 µm Y-27632 (Y27) or the vehicle control (deionized water). Scale bar, 100 µm. (D) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces treated with 20 µm Y-27632 or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. For D, each outline represents a 4-s interval. (E) Quantification of the percentage of blebbing cells ( N = 3, n = 28–31 fields of view) and the number of blebs per minute per cell ( N = 3, n = 36 cells) for cells treated with 20 µm Y-27632 or the vehicle control. (F) Quantification of the percentage of blebbing cells in cells treated with 25 µm Blebb or the vehicle control ( N = 3, n = 29 fields of view). (G) Normalized GTP-loaded RhoA measured using absorbance at 490 nm for cells treated with 20 µm Y-27632 or the vehicle control ( N = 4). Bar graphs denote the mean ± SEM. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Activity Assay, Membrane, Labeling, Control

    Representative time lapse of an MDA-MB-231 Vcl KO cell expressing LifeAct (red) and stained with 20 nM MemGlow (green), a membrane dye. The time lapse was acquired on a Zeiss LSM800 confocal microscope at 5-s intervals and represents the MDA-MB-231 Vcl KO cell pictured in . Scale bar, 10 µm. Playback, 3 fps.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative time lapse of an MDA-MB-231 Vcl KO cell expressing LifeAct (red) and stained with 20 nM MemGlow (green), a membrane dye. The time lapse was acquired on a Zeiss LSM800 confocal microscope at 5-s intervals and represents the MDA-MB-231 Vcl KO cell pictured in . Scale bar, 10 µm. Playback, 3 fps.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Expressing, Staining, Membrane, Microscopy

    Representative time lapse of an MDA-MB-231 cell expressing LifeAct (red) and stained with 20 nM MemGlow (green), a membrane dye. The time lapse was acquired on a Zeiss LSM800 confocal microscope at 5-s intervals and represents the MDA-MB-231 cell pictured in . Scale bar, 10 µm. Playback, 3 fps.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative time lapse of an MDA-MB-231 cell expressing LifeAct (red) and stained with 20 nM MemGlow (green), a membrane dye. The time lapse was acquired on a Zeiss LSM800 confocal microscope at 5-s intervals and represents the MDA-MB-231 cell pictured in . Scale bar, 10 µm. Playback, 3 fps.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Expressing, Staining, Membrane, Microscopy

    Representative phase-contrast time lapse of MDA-MB-231 cells moving in 2D, treated with vehicle control for Y-27632 (deionized water) for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative phase-contrast time lapse of MDA-MB-231 cells moving in 2D, treated with vehicle control for Y-27632 (deionized water) for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Control, Inverted Microscopy

    Representative phase-contrast time lapse of MDA-MB-231 Vcl KO cells moving in 2D, treated with vehicle control for Y-27632 (deionized water) for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative phase-contrast time lapse of MDA-MB-231 Vcl KO cells moving in 2D, treated with vehicle control for Y-27632 (deionized water) for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Control, Inverted Microscopy

    Representative phase-contrast time lapse of MDA-MB-231 cells moving in 2D, treated with 20 µM Y-27632 for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative phase-contrast time lapse of MDA-MB-231 cells moving in 2D, treated with 20 µM Y-27632 for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Inverted Microscopy

    Representative phase-contrast time lapse of MDA-MB-231 Vcl KO cells moving in 2D, treated with 20 µM Y-27632 for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Representative phase-contrast time lapse of MDA-MB-231 Vcl KO cells moving in 2D, treated with 20 µM Y-27632 for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Inverted Microscopy

    Metabolic inhibition of Vcl KO cells reduces cell blebbing, and Rho activation of parental MDA-MB-231 cells increases cell blebbing and bioenergetics. (A and B) Representative (A) phase-contrast images (scale bar, 100 µm) and (B) color-coded time lapses of Vcl KO cells treated with oligomycin, iodoacetate, or the vehicle control for each drug. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (C) Percentage of blebbing cells treated with oligomycin ( N = 3, n = 33–34 fields of view), iodoacetate ( N = 3, n = 31 fields of view), or the vehicle and the number of blebs per cell per minute for Vcl KO cells treated with oligomycin ( N = 3, n = 32–33 cells), iodoacetate ( N = 3, n = 32–34 cells), or the vehicle control. (D) Cell spread area of Vcl KO cells treated with oligomycin ( N = 3, n = 92–97 cells), iodoacetate ( N = 3, n = 101–137 cells), or the vehicle control, and the aspect ratio of Vcl KO cells treated with oligomycin ( N = 3, n = 90–91 cells), iodoacetate ( N = 3, n = 94–134 cells), or the vehicle control. (E and F) Representative (E) phase-contrast images (scale bar, 100 µm) and (F) color-coded time lapses of parental MDA cells treated with Rho activator or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (G) Percentage of blebbing cells ( N = 3, n = 36 fields of view) or number of blebs per cell per minute ( N = 3, n = 36 cells) for MDA cells treated with Rho activator or the vehicle control. (H) Mean intensity of 2-NBDG in parental MDA cells treated with Rho activator or the vehicle control ( N = 3, n = 90–97 cells). (I) Mean intensity of TMRM in parental MDA cells treated with Rho activator or the vehicle control ( N = 3, n = 61–62 cells). (J) Cell spread area ( N = 3, n = 106–140 cells) and aspect ratio ( N = 3, n = 106–140 cells) of parental MDA cells treated with the Rho activator compared with Vcl KO cells. (K) Cell spread area of parental MDA cells being treated with 20 µm oligomycin or the vehicle control over time after trypsinization ( N = 3, n = 74–89 cells). (L) Cell spread area of parental MDA cells being treated with 10 µm iodoacetate or the vehicle control over time after trypsinization ( N = 3, n = 72–87 cells). For K and L, time of 0 h indicates when cells are initially seeded on tissue culture surface and not yet adhered or spread. Bar graphs denote the mean ± SEM. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). XY plots show the mean ± SEM. ns = not significant, **P < 0.01, ****P < 0.0001.

    Journal: The Journal of Cell Biology

    Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

    doi: 10.1083/jcb.202504025

    Figure Lengend Snippet: Metabolic inhibition of Vcl KO cells reduces cell blebbing, and Rho activation of parental MDA-MB-231 cells increases cell blebbing and bioenergetics. (A and B) Representative (A) phase-contrast images (scale bar, 100 µm) and (B) color-coded time lapses of Vcl KO cells treated with oligomycin, iodoacetate, or the vehicle control for each drug. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (C) Percentage of blebbing cells treated with oligomycin ( N = 3, n = 33–34 fields of view), iodoacetate ( N = 3, n = 31 fields of view), or the vehicle and the number of blebs per cell per minute for Vcl KO cells treated with oligomycin ( N = 3, n = 32–33 cells), iodoacetate ( N = 3, n = 32–34 cells), or the vehicle control. (D) Cell spread area of Vcl KO cells treated with oligomycin ( N = 3, n = 92–97 cells), iodoacetate ( N = 3, n = 101–137 cells), or the vehicle control, and the aspect ratio of Vcl KO cells treated with oligomycin ( N = 3, n = 90–91 cells), iodoacetate ( N = 3, n = 94–134 cells), or the vehicle control. (E and F) Representative (E) phase-contrast images (scale bar, 100 µm) and (F) color-coded time lapses of parental MDA cells treated with Rho activator or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (G) Percentage of blebbing cells ( N = 3, n = 36 fields of view) or number of blebs per cell per minute ( N = 3, n = 36 cells) for MDA cells treated with Rho activator or the vehicle control. (H) Mean intensity of 2-NBDG in parental MDA cells treated with Rho activator or the vehicle control ( N = 3, n = 90–97 cells). (I) Mean intensity of TMRM in parental MDA cells treated with Rho activator or the vehicle control ( N = 3, n = 61–62 cells). (J) Cell spread area ( N = 3, n = 106–140 cells) and aspect ratio ( N = 3, n = 106–140 cells) of parental MDA cells treated with the Rho activator compared with Vcl KO cells. (K) Cell spread area of parental MDA cells being treated with 20 µm oligomycin or the vehicle control over time after trypsinization ( N = 3, n = 74–89 cells). (L) Cell spread area of parental MDA cells being treated with 10 µm iodoacetate or the vehicle control over time after trypsinization ( N = 3, n = 72–87 cells). For K and L, time of 0 h indicates when cells are initially seeded on tissue culture surface and not yet adhered or spread. Bar graphs denote the mean ± SEM. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). XY plots show the mean ± SEM. ns = not significant, **P < 0.01, ****P < 0.0001.

    Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

    Techniques: Inhibition, Activation Assay, Control, Whisker Assay