Figure 5.
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.

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.

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