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: MDA-MB-231 cells stably co-expressing PercevalHR (#49083; Addgene) and pHRed (#31473; Addgene) probes, gifts from Gary Yellen (Harvard Medical School, Boston, MA, USA), were transfected with 50 pmol/μl nontargeting scrambled control siRNA (AM4611; Thermo Fisher Scientific) or siRNA targeting Vcl (AM51331 [siRNA ID: 108455] and AM16708 [siRNA ID: 108453]; Thermo Fisher Scientific) using Lipofectamine 2000 (11668030; Thermo Fisher Scientific) and Opti-MEM transfection medium (31985062; Thermo Fisher Scientific) according to the manufacturer’s instructions.
Techniques: Western Blot, Expressing, Transfection, Knockdown, Control, Staining, Whisker Assay
