Journal: APL Bioengineering

Article Title: Survivin modulates stiffness-induced vascular smooth muscle cell motility

doi: 10.1063/5.0252766

Figure Lengend Snippet: Survivin inhibition reduces collective cell migration. VSMCs plated on cell culture plates were treated with various doses of YM155 (c), 10 µg/ml of Mitomycin C (e), or DMSO (a vehicle control). (a) Total cell lysates were collected for immunoblotting. (b) The graphs show the expression of survivin in VSMCs treated with YM155, normalized to that in VSMCs treated with DMSO. n = 5 independent biological replicates. (c) and (e) A scratch-wound assay was performed, and images were captured at 0, 12, and 24 h post-treatment. n = 5–12 independent biological replicates (c) and n = 5 independent biological replicates (e). (d) and (f) Wound closure (%) at 12 and 24 h was calculated using the formula: [(Initial wound size) − (wound size at 12 or 24 h)]/(wound size at 12 or 24 h) × 100. ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: VSMCs were plated on plastic cell culture plates, glass coverslips, or soft and stiff hydrogels in media containing 10% serum with varying doses (0.1, 0.5, 1, or 2 µM) of YM155 (survivin inhibitor; Cat. No. 11490, Cayman Chemical), 10 µg/ml Mitomycin C (proliferation inhibitor; Cat. No. BML-GR311-0002, Enzo Life Sciences), or dimethyl sulfoxide (DMSO; Cat. No. D8418, Sigma-Aldrich) as a vehicle control.

Techniques: Inhibition, Migration, Cell Culture, Control, Western Blot, Expressing, Scratch Wound Assay Assay

Journal: APL Bioengineering

Article Title: Survivin modulates stiffness-induced vascular smooth muscle cell motility

doi: 10.1063/5.0252766

Figure Lengend Snippet: Survivin inhibition decreases single cell migration. (a) VMSCs were sparsely plated on cell culture plates and treated with either YM155 or DMSO for 4 h. Total cell lysates were collected for immunoblotting. (b) The graph shows the expression of survivin in VSMCs treated with varying concentrations of YM155, normalized to that of VSMCs treated with DMSO. n = 4 independent biological replicates. (c)–(e) VSMCs were plated for time-lapse imaging. Manual tracking of these cells was conducted to obtain single-cell trajectories (c) and average cell velocities (d). 49 cells (DMSO), 45 cells (0.1 µM YM155), 42 cells (0.5 µM YM155), 44 cells (1 µM YM155), and 45 cells (2 µM YM155) were analyzed from n = 3 independent biological replicates. (e) Sequences of images from a set of representative time-lapse experiments (arrow-direction of protrusion). * p < 0.05, ** p < 0.01, *** p < 0.001 . ns, not significant.

Article Snippet: VSMCs were plated on plastic cell culture plates, glass coverslips, or soft and stiff hydrogels in media containing 10% serum with varying doses (0.1, 0.5, 1, or 2 µM) of YM155 (survivin inhibitor; Cat. No. 11490, Cayman Chemical), 10 µg/ml Mitomycin C (proliferation inhibitor; Cat. No. BML-GR311-0002, Enzo Life Sciences), or dimethyl sulfoxide (DMSO; Cat. No. D8418, Sigma-Aldrich) as a vehicle control.

Techniques: Inhibition, Migration, Cell Culture, Western Blot, Expressing, Imaging

Journal: APL Bioengineering

Article Title: Survivin modulates stiffness-induced vascular smooth muscle cell motility

doi: 10.1063/5.0252766

Figure Lengend Snippet: Pharmacological inhibition of survivin reduces stiffness-dependent migration. (a) VSMCs were sparsely seeded on fibronectin-coated soft and stiff hydrogels and treated with YM155 or DMSO for 4 h, followed by collection of total lysates for immunoblotting. (b) The graph shows survivin expression in cells treated with varying concentrations of YM155, normalized to the expression in VSMCs on stiff hydrogels treated with DMSO. n = 4 independent biological replicates. (c)–(e) VSMCs were plated for time-lapse video microscopy, and manual tracking of these cells was conducted to obtain single-cell trajectories (c) and average cell velocities (d). 35 cells (soft-DMSO), 36 cells (stiff-DMSO), 38 cells (stiff-0.1 µM YM155), 39 cells (stiff-0.5 µM YM155), 35 cells (stiff-1 µM YM155), and 35 cells (stiff- 2 µM YM155) were analyzed from n = 3 independent biological replicates. (e) Sequences of images from a set of representative time-lapse experiments (arrow-direction of protrusion). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: VSMCs were plated on plastic cell culture plates, glass coverslips, or soft and stiff hydrogels in media containing 10% serum with varying doses (0.1, 0.5, 1, or 2 µM) of YM155 (survivin inhibitor; Cat. No. 11490, Cayman Chemical), 10 µg/ml Mitomycin C (proliferation inhibitor; Cat. No. BML-GR311-0002, Enzo Life Sciences), or dimethyl sulfoxide (DMSO; Cat. No. D8418, Sigma-Aldrich) as a vehicle control.

Techniques: Inhibition, Migration, Western Blot, Expressing, Microscopy

Journal: APL Bioengineering

Article Title: Survivin modulates stiffness-induced vascular smooth muscle cell motility

doi: 10.1063/5.0252766

Figure Lengend Snippet: Survivin regulates FAK phosphorylation, actin organization, and stress fiber formation. (a) VMSCs were sparsely seeded on fibronectin-coated soft and stiff hydrogels and treated with either YM155 or DMSO. Cells were stained with DAPI, phospho-FAK (Tyr397) antibody and Alexa Fluor 647-phalloidin. Fluorescence images were acquired using a spinning disk confocal microscope with a 63× oil-immersion objective. The outlined boxes indicate the regions shown in the magnified insets. (b) A sum of slices projection of each cell was generated using Fiji/ImageJ, and FAK pY397 clusters were manually counted. 37 cells (soft-DMSO), 44 cells (stiff-DMSO), 33 cells (stiff-0.1 µM YM155), and 37 cells (stiff-2µM YM155) were analyzed from n = 3 independent biological replicates. * p < 0.05, ** p < 0.01.

Article Snippet: VSMCs were plated on plastic cell culture plates, glass coverslips, or soft and stiff hydrogels in media containing 10% serum with varying doses (0.1, 0.5, 1, or 2 µM) of YM155 (survivin inhibitor; Cat. No. 11490, Cayman Chemical), 10 µg/ml Mitomycin C (proliferation inhibitor; Cat. No. BML-GR311-0002, Enzo Life Sciences), or dimethyl sulfoxide (DMSO; Cat. No. D8418, Sigma-Aldrich) as a vehicle control.

Techniques: Phospho-proteomics, Staining, Fluorescence, Microscopy, Generated