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. 2015 Jan 1;128(1):50–60. doi: 10.1242/jcs.151647

Fig. 2.

Fig. 2.

p190 acts as a Rho GAP in the cytokinesis furrow. (A) p190-depleted cells have increased RhoA-GTP levels in the cytokinetic furrow. RhoA-GTP levels were measured with the colorimetric RhoA G-LISA assay. Active RhoA-GTP protein was used as a positive control and set at 100%. Results are expressed as the mean±s.e.m. percentage of RhoA-GTP, n = 4. *P<0.05 (Student's t-test) as compared to cytokinetic cells. (B) Confocal images of HeLa cells during cytokinesis stained for RhoA and tubulin 48 hours after transfection of siRNA targeted against LacZ (control siRNA) or p190. Images shown are representative of n>20. Scale bars: 7 µm. (B′) Quantification of RhoA intensities at the cytokinetic furrow (CF). n>20, *P = 0.0002 (Student's t-test). The box represents the 25–75th percentiles, and indicates the median. The whiskers show the range. (C) Confocal images of pMLC II in wild-type p190 doxycycline-inducible HeLa cells during cytokinesis and at 48 hours after treatment with control siRNA (LacZ), p190 siRNA and p190 siRNA with doxycycline treatment. Fixed cells were stained with anti-pSer19 MLC II antibody. Images shown are representative of n>20. Scale bars: 4.5 µm. (C′) Quantification of pMLC at the cytokinetic furrow. Box-and-whisker plot quantifying the ratio of intensity of pMLC II at the cytokinetic furrow and outside region of cytokinetic furrow. n>2 with >20 cells/experiment. *, **P<0.005 as compared to LacZ-siRNA-treated cells. (D) A GAP mutant of p190 does not rescue p190-silencing-induced multinucleation. Cells were depleted of p190 and simultaneously were treated with 1 µg/ml of doxycycline to re-express the doxycycline-inducible GAP mutant of p190. Failed cytokinesis was scored as the percentage of binucleated or multinucleated interphase cells (n≥300). Results are mean±s.d. from three independent experiments. *P<0.0001 for both p190 siRNA-treated cells and cells rescued by p190 GAP mutant (p190 R1283A) compared to control. Note that the experiment in Fig. 1B was performed under identical conditions and at the same time, and acts as a control for rescue with wild-type protein. (E) Immunoblot to mesure relative p190 levels. HeLa cells were depleted of endogenous p190 and a GAP mutant of p190 (p190 R1283A) was expressed by the addition of doxycyclin where indicated. Cell lysates were prepared for immunoblot analysis 48 hours post-transfection.