6A). and correlated with an increased presence of mitotic spindle abnormalities in the -actin depleted cells. Collectively, these results demonstrate a previously unknown role for -actin in regulating centrosome function, chromosome alignment and maintenance of mitotic spindle integrity. statistically significant between the drug treated cells and its corresponding drug free cells in G0/G1 and G2/M cell cycle phases. statistically significant between the drug treated cells and its corresponding drug free cells in S phase. statistically significant between the drug treated cells and its corresponding drug free cells in sub-G1. Open in a separate window Physique 5. Partial depletion of -actin inhibits Aurora kinase A inhibitor, MLN8273, induced G2/M cell cycle checkpoint in SH-EP cells. Cell cycle distribution in SH-EP GFP–Itubulin cells transfected with either the control (A) or -actin siRNA (B) then treated with increasing concentrations of MLN8237, for either 22 (A(i) and B(i)) or 70?h (A(ii) and B(ii)). Data are mean SEM of 3 impartial experiments.statistically significant between the drug treated cells and its corresponding drug free cells in G0/G1 and G2/M cell cycle phases. statistically significant between the drug treated cells and its corresponding drug free cells in S phase. statistically significant between the drug treated cells and its corresponding drug free cells in sub-G1. Depletion of -actin leads to enhanced centrosome amplification The results showing that -actin is required for drug-induced mitotic arrest, raised the possibility that -actin may be involved in regulating mitotic progression. To address this, we PD173074 initially investigated whether depletion of -actin affects centrosome amplification since a correct number of centrosomes are essential for normal mitosis to occur. Extra PD173074 centrosomes were frequently observed in SH-EP cells treated with -actin siRNA (7.3%, a 2.1-fold increase) compared to the control siRNA (3.4%) (Fig. 6A). Furthermore, a pattern toward increased number of extra centrosomes was also obtained in MCF-7 cells transfected with -actin PD173074 siRNA, where a 2.1-fold increase in centrosome amplification was observed, compared to the control siRNA (Fig. 6B). These results were further confirmed in SH-EP cells transfected with either of 2 additional -actin siRNAs, duplex 1 and duplex 2 (a 3.0-fold Rabbit polyclonal to FosB.The Fos gene family consists of 4 members: FOS, FOSB, FOSL1, and FOSL2.These genes encode leucine zipper proteins that can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP-1. and 2.5-fold increase in centrosome amplification was observed, respectively) (Fig. S7A). Open in a separate window Physique PD173074 6. Enhanced centrosome amplification in cancer cells due to -actin depletion. Quantification of centrosome numbers in the control and -actin siRNA transfected SH-EP (A), MCF-7 (B) and MRC-5 cells (C) were analyzed by co-staining the cells with -tubulin and -tubulin. Confocal images were acquired and then centrosome number was scored in interphase cells as 1, 2 or more than 2 centrosomes. n = the total number of interphase cells analyzed from 4 impartial experiments. = 0.0017) compared to control siRNA (6.5%) (Fig. 8A-B). The majority of the observed spindle defects were bipolar spindles with uncongressed chromosomes (see arrow in Fig. 8A(iii)), with a few multipolar spindles (Fig. 8A(iv)) and anaphase bridges (Fig. 8A(v)). Comparable types of spindle defects were detected in the control cells, but occurred at a much lower frequency compared to the -actin-depleted cells (Fig. 8B). Increased formation of spindle abnormalities was also observed in MCF-7 cells treated with -actin siRNA (12.6%, 2.16-fold increase, = 0.0254) compared to the control siRNA (5.8%) (Fig. 8C) and was also confirmed with 2 additional -actin siRNAs in both SH-EP (Fig. S7B) and MCF-7 cells (Fig. S7C). The two additional siRNAs, -actin siRNA duplex 1 and 2.