Transient treatment with ROCK inhibitors is sufficient to promote GSC-like cell expansion.

<p>The cells were treated with no inhibitor, with continuous exposure to the ROCK inhibitor (45 μM Y-27632 or 10 μM fasudil), or with transient exposure to the ROCK inhibitor (45 μM Y-27632 or 10 μM fasudil). (A) Representative micrographs of each experimental group on Day 3 (Scale bars = 100 μm). (B) The sphere diameter and number of spheres were analyzed for all experimental groups on Day 3 (mean ± SE; <i>n</i> = 100; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001). The number of spheres per field of view in each experimental group were also quantified from the micrographs (mean ± SE; <i>n</i> = 20; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001).</p

ROCK inhibitors enhance GSC-like stemness.

<p>(A) The clonogenicity of the cells was quantified using limiting dilution assay (steeper slope and lower value in x-intercept indicates increased clonogenic potential and stemness). The cells treated with 45 μM Y-27632 or 10 μM fasudil required fewer cells to form spheres indicating increased number of GSC-like cell than control. (B) The glioblastoma cells were grown as tumorspheres in two concentrations of Y-27632 (0 and 45μM) or two concentrations of fasudil (0 and 10 μM) for three days. Using flow cytometry, the percentage of the total population expressing the GSC marker SOX2 was quantified.</p

Knockdown of ROCK2 shows similar behavior to Y-27632 and Fasudil.

<p>U87-MG cells were transfected with ROCK2 siRNA and grown as tumorspheres for 3 days. The cells’ ability to form spheres was analyzed, and qRT-PCR was performed to confirm the success of the transfection. A) Representative micrographs of each experimental group on Day 1 (Scale bar = 100 μm). B) The sphere diameter (mean ± SE; <i>n</i> = 100; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001) and number of spheres per field of view were analyzed for all experimental groups on Day 1 (mean ± SE; <i>n</i> = 20; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001). C) qRT-PCR was performed on the transfected cells to confirm their gene expression levels of <i>ROCK2</i>, <i>CASP3</i>, and <i>CASP7</i>. Expression is reported as percentage of that of negative control (mean ± SE; <i>n</i> = 3; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001).</p

ROCK inhibitors enhance GBM tumorsphere formation.

<p>The glioblastoma cells were grown as tumorspheres in two concentrations of Y-27632 (0 and 45 μM) or two concentrations of fasudil (0 and 10μM) for 6 days. (A) Sample micrographs of each experimental group on Day 6 (Scale bars = 100 μm). (B) The sphere diameter and number of spheres were analyzed for all experimental groups on Day 3 (mean ± SE; <i>n</i> = 100). It was found that the sphere diameter stayed relatively consistent between the experimental groups. The number of spheres per field of view in each experimental group were also quantified from the micrographs (mean ± SE; <i>n</i> = 20; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001).</p

ROCK inhibitors are not toxic to GBM tumorspheres at low concentrations.

<p>The toxicities of Y-27632 and fasudil were measured using a water-soluble tetrazolium assay (WST-8 Cell Counting Kit 8). U87-MG, JX12, and SMC448 cells were exposed to varying concentrations of Y-27632 or fasudil for 48 hours. Cell viability was measured relative to 0 μM control (<i>n</i> = 10).</p

ROCK inhibitors protect GBM tumorspheres from apoptosis.

<p>Flow cytometry was used to quantify the late-stage apoptotic cells (Annexin V⁺/PI⁺) immediately after trituration. The cells that were treated with 45μM Y-27632 or 10 μM fasudil had decreased number of late-stage apoptotic cells in U87-MG, JX12, and SMC448 cell lines, indicating that the ROCK inhibitors Y-27632 and fasudil inhibited apoptosis in glioblastoma cells.</p