Journal: International Journal of Molecular Sciences

Article Title: Inhibitors of the VEGF Receptor Suppress HeLa S3 Cell Proliferation via Misalignment of Chromosomes and Rotation of the Mitotic Spindle, Causing a Delay in M-Phase Progression

doi: 10.3390/ijms19124014

Figure Lengend Snippet: Vascular endothelial growth factor receptor (VEGFR) inhibitors suppress cell proliferation. ( A ) HeLa S3 cells were treated with A83-01, SU4312, and Ki8751 at the indicated concentrations for 2 days, and viable cells were determined by monitoring the absorbance of the formazan at 450 nm. Relative values are shown as a ratio of absorbance to solvent control (di-methyl sulfoxide, DMSO) using the mean ± S.D., calculated from three independent experiments. IC 50 was calculated in A83-01–, Ki8751–treated cells in each experiment, and the mean ± S.D. was shown in the graph. Asterisks indicate statistical significance (* p < 0.05; ** p < 0.01; NS, not significant), calculated using Scheffe’s F test. ( B ) Cells were cultured without serum for 1 day and then pretreated with the indicated inhibitors or DMSO as a solvent control for 30 min. Then, serum was added into the culture, and cells were continuously treated with inhibitors for 30 min. Whole cell lysates were analyzed using Western blot analysis with anti-phospho-Akt (pSer473) and anti-α-tubulin antibodies. Phosphorylation of Akt was quantified by measuring the signal intensity of the bands. The ratios of signal intensity of phosphorylated band of Akt to that of α-tubulin are shown as the mean ± S.D., calculated from three independent experiments. Asterisk indicates statistical significance (* p < 0.05; NS, not significant), calculated using Scheffe’s F test. ( C ) Cells were treated with 20 µM A83-01, 20 µM SU4312, 20 µM Ki8751, and 4 µM Adriamycin (ADR) for 24 h and then fixed and stained for DNA (red), α-tubulin (green), and cleaved caspase-3 (blue). Scale bar, 100 µm. The number of cells with multinuclei or micronuclei was counted and is shown as the mean ± S.D., calculated from three independent experiments ( n > 155 in each treatment). Asterisks indicate statistical significance (** p < 0.01), calculated using Scheffe’s F test. ( D ) (Left), cells were treated with 20 µM A83-01, 20 µM SU4312, 10 µM Ki8751 and 4 µM ADR for 24 h, and the lysate was prepared and analyzed for cleaved caspase-3. (Right), cells were treated with 4 µM ADR for 48 h, and viable cells were determined as shown in (A). Relative values are shown as a ratio using the mean ± S.D., calculated from three independent experiments. Asterisks indicate statistical significance (** p < 0.01), calculated using Student’s t -test.

Article Snippet: The VEGFR inhibitors SU4312 (Sigma-Aldrich, St. Louis, MO, USA), A83-01 (StressMarq Bioscience Inc., Victoria, BC, Canada), and Ki8751 (Selleck Chemicals, Houston, TX, USA) were used in the present study.

Techniques: Cell Culture, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Inhibitors of the VEGF Receptor Suppress HeLa S3 Cell Proliferation via Misalignment of Chromosomes and Rotation of the Mitotic Spindle, Causing a Delay in M-Phase Progression

doi: 10.3390/ijms19124014

Figure Lengend Snippet: VEGFR inhibitors cause aberrant M phase progression. Cells were treated with 6 µM RO-3306 for 20 h and released in the presence of DMSO, 3 µM A83-01, 10 µM SU4312, or 1 µM Ki8751, together with 0.1 µM Hoechst 33342 to visualize DNA. Mitotic progression was monitored every 5 min for 3 h by time-lapse imaging. ( A ) Representative images of cells exhibiting normal M phase progression, misalignment of chromosomes, and rotation of the mitotic spindle are shown. Scale bars, 10 µm. ( B ) Based on the time-lapse images shown in ( A ), the duration of each mitotic phase (prophase and prometaphase(P/PM, light green), metaphase (M, red), anaphase and telophase (A/T, blue)), misalignment of chromosomes (deep green), and rotation of the mitotic spindle (orange) in individual cells are shown (DMSO, n = 34; A83-01, n = 38; SU4312, n = 35; Ki8751, n = 37). ( C ) The percentages of mitotic cells (black), cells in prophase and prometaphase (P/PM, green), metaphase (M, red), and anaphase and telophase (A/T, blue) at the indicated times are shown.

Article Snippet: The VEGFR inhibitors SU4312 (Sigma-Aldrich, St. Louis, MO, USA), A83-01 (StressMarq Bioscience Inc., Victoria, BC, Canada), and Ki8751 (Selleck Chemicals, Houston, TX, USA) were used in the present study.

Techniques: Imaging

Journal: International Journal of Molecular Sciences

Article Title: Inhibitors of the VEGF Receptor Suppress HeLa S3 Cell Proliferation via Misalignment of Chromosomes and Rotation of the Mitotic Spindle, Causing a Delay in M-Phase Progression

doi: 10.3390/ijms19124014

Figure Lengend Snippet: Rotation of mitotic spindle in A83-01-treated cells. ( A ) After incubation with RO-3306 for 20 h, cells were released in the presence of 3 µM A83-01 for 1 h, fixed, and stained for α-tubulin, γ-tubulin and DNA. (Left), z –stack images were acquired by using a confocal microscopy, and one or two focal planes ( x - y images) were shown. (Right), the x – z projections from the z –stack images of 25 focal planes (1 µm apart). Arrows indicate the positions of centrosomes. Scale bars, 10 µm. ( B ) After incubation with RO-3306, cells were released in the presence of 0.1 µM Hoechst 33342 with 10 µM MG-132 or 3 µM A83-01. MG-132 or A83-01 was added into the culture at the time of release or at 30 min after the release, respectively. Then, mitotic progression was monitored every 5 min by time-lapse imaging until 3 h after the release. Fluorescence of Hoechst 33342 and bright field images are shown. Scale bars, 10 µm.

Article Snippet: The VEGFR inhibitors SU4312 (Sigma-Aldrich, St. Louis, MO, USA), A83-01 (StressMarq Bioscience Inc., Victoria, BC, Canada), and Ki8751 (Selleck Chemicals, Houston, TX, USA) were used in the present study.

Techniques: Incubation, Staining, Confocal Microscopy, Imaging, Fluorescence