Fig. 2.

Ensa KD increases S-phase length. a HeLa cells treated with SC or Ensa siRNA were blocked in G1/S by thymidine and released in fresh medium. Samples were taken at 4, 6, 8 and 10 h after release and processed by FACS to follow S-phase progression. b As for a except that U2OS cells were used and released at 4, 9, 14 and 17 h. c Scheme representing the experiment in which HeLa cells were treated with SC or Ensa siRNA for 48 h, pulsed with 10 µM EdU for 30 min and, after washing, incubated in fresh medium containing 5 µM thymidine for 2, 4, 6, 8 and 10 h. After a second pulse of 30 min with 10 µM, BrdU cells were fixed for immunofluorescence. d EdU/BrdU double staining of siSC and siEnsa-treated cells at 10 h after thymidine release is shown. EdU-positive cells are labelled in magenta and BrdU-positive cells in cyan. Resulting white co-localisation are cells still in S phase. Scale bar, 20 μm. e The % of EdU/BrdU-positive cells in d was counted and represented as a bar graph of the mean value ± standard deviation. A minimum of 80 cells was counted in each time point. f HeLa cells were or not (CT) transfected with increasing doses of siEnsa (12.5, 25 and 50 nM) and 24 h later synchronised in S phase by thymidine block. Cells were then released and followed by time-lapse microscopy. Time after thymidine release and cell rounding (G2/M transition) for each cell at each siRNA concentration was recorded and represented as a box-and-whiskers diagram showing median and minimum to maximum values. A minimum of 50 cells was counted per condition. g Asynchronous control or HeLa cells stably expressing a siEnsa-resistant transgene of human Ensa (R-Ensa) were transfected with siEnsa1 or with SC siRNA (only for parental cells). Cells were released 24 h later, recovered and used for FACS analysis. h As for g, except that cells were blocked in G1–S 24 h after transfection by thymidine and 9 h after release used for FACs analysis