FIGURE 5.

Modulation of the cellular response to paclitaxel (Tx) treatment by hGTSE-1, in a p21-dependent manner. A, HeLa cells transfected with a control (siCONT) or hGTSE-1 (sihGTSE-1) siRNA for 36 h were treated with paclitaxel (0.5 μm) for an additional 24 h (left panel). JPIC/U cells (middle panel) or JPIC/H cells (right panel) were treated or without ponasterone A (pon A and −, respectively) for 16 h followed by addition of paclitaxel (0,5 μm) for another 24 h. The upper bands of hGTSE-1 and p21 detected in paclitaxel-treated cells correspond to phosphorylated forms seen in mitotic cells (5, 13). B, JPIC/UP and EPIC/UP cells were treated with (pon A) or without (−) ponasterone A for 16 h, followed by addition of paclitaxel (1 μm) for another 24 h. C, colony formation assays of cells treated as in A and B. JPIC/U and EPIC/UP cells were treated with 1 μm paclitaxel for 24 h. JPIC/UP cells behave as JPIC/U and are not shown. JPIC/H cells were treated with 0.2 μm paclitaxel for 24 h. HeLa cells were treated with 0,05 μm paclitaxel for 24 h. Histograms represent the mean ratio between paclitaxel-treated siCONT:sihGTSE-1 (HeLa) and ponasterone A:noninduced cells (−) (JPIC/U, EPIC/UP, and JPIC/H). D, JPIC/U cells were treated as in A but after transfecting a control (siCONT) or p21 (sip21) siRNAs 36 h before ponasterone A treatment. All immunoblots were performed with antibodies against hGTSE-1, p21, cleaved caspase-3, and actin as loading control. Numbers at the bottom of the immunoblots indicate representative values of the sub-G₁ DNA content calculated trough normalization by biological background subtraction noise (see ”Experimental Procedures“).