Cdc6 depletion reverses the PTX-induced Cdk1 inactivation in Hela cells.

<p>Hela cells were treated with PTX (30 nM) combination with or without Cdc6 RNAi or NCTD (30 μM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162633#pone.0162633.g007" target="_blank">Fig 7</a>. Cdc6, pCdk1 and Rad 21 were examined by Western Blotting. GAPDH was used as loading control. The protein levels are expressed as optical density fold difference related to GAPDH (relative OD). Three independent experiments were performed, *<i>P</i><0.05 as compared to PTX group.</p

Cdc6 depletion inhibits mitotic slippage in synchronized cells.

<p>The synchronized cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162633#pone.0162633.g004" target="_blank">Fig 4</a> were collected after released for 24, 48 and 72h. (A) Cells were stained with Giemsa dye and the percentage of slippage cells is presented in (right panel). (B) Mitotic cells were examined by pH3 immunostaining and the mitotic index was calculated as pH3 cells/DAPI cells ×100% (below panel).</p

Mitotic slippage arises after PTX treatment.

<p>(A) HepG2 cell were treated with indicative concentration of PTX for 3 days or (B) with 30 nM PTX for 1 to 7 days. Cells were stained with Giemsa dye and viewed under bright field microscope. Representative photomicrographs are shown. The percentage of slippage cells is presented in below panel; (C) Cell cycle progression of (B) was analyzed by FACS; percentage of G2/M (upper) and G1 (below) are presented respectively in right panel. All of the experiments were performed in three independent experiments, *<i>P</i><0.05.</p

Cdc6 contributes to PTX-induced mitotic slippage in synchronized cells.

<p>The synchronized cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162633#pone.0162633.g005" target="_blank">Fig 5</a> were collected to extract total proteins. Cdc6 and pCdk1 were examined by Western Blotting. The protein levels are expressed as relative OD. All of the experiments were performed in three independent experiments, *<i>P</i><0.05 as compared to PTX group.</p

Cdc6 depletion inhibits PTX-induced mitotic slippage in Hela cells.

<p>(A) Hela cells were treated with PTX (30 nM) for 1 to 5 days, NCTD (30 μM) was added to replace PTX after 3 days PTX treatment. Cdc6 RNAi was transfected after PTX treatment for 3 days. Cells were stained with Giemsa dye and the percentage of slippage cells is calculated (right panel). (B) Cell viability after PTX treatment combined with or without NCTD (30 μM) for 7 days was valued by typan blue assay. All of the experiments were performed in three independent experiments, *<i>P</i><0.05 as compared to PTX group.</p

Cdc6 depletion inhibits PTX-induced mitotic slippage and reverses PTX resistance.

<p>(A) HepG2 cells were treated with combination with or without Cdc6 RNAi or NCTD (30 μM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162633#pone.0162633.g002" target="_blank">Fig 2</a>. At day 7, Cells were stained with Giemsa dye and the percentage of slippage cells is presented (right panel). (B) Cell cycle was analyzed by FACS. G2/M (upper) and G1 (below) percentage were presented in right panel. (C) Cell viability after PTX treatment combined with or without NCTD (30 μM) for 10 days was valued by typan blue assay. All of the experiments were performed in three independent experiments, *<i>P</i><0.05 as compared to PTX group.</p

Cdc6 depletion inhibits mitotic exit in synchronized cells.

<p>HepG2 cells were synchronized by double thymidine block, and then released into fresh medium containing PTX (30 nM) or PTX (30nM) + NCTD (30 μM). For Cdc6 RNAi, the transfection was performed after first thymidine block. Cell cycle profiles were analyzed by FACS after released for indicative time. G2/M (left) and G1 (right) percentage are presented in below panel. Three independent experiments were performed.</p

Cdc6 contributes to PTX-induced mitotic slippage by inactivation of Cdk1.

<p>(A) HepG2 cells were transfected with siRNA targeting cdc6 mRNA for 24h, the inhibitory effect was examined by Western Blotting. (B) HepG2 cells were treated with PTX (30 nM) for 4 and 7 days, NCTD (30 μM) was added to replace PTX after 4 days treatment. Cdc6 RNAi was transfected after PTX treatment for 4 days. Cdc6, pCdk1 and Rad 21 were examined by Western Blotting. GAPDH was used as loading control. The protein levels are expressed as optical density fold difference related to GAPDH (relative OD). Three independent experiments were performed, *<i>P</i><0.05 as compared to PTX group.</p