Fig. 3. Depletion of centrosomal proteins does not necessarily cause cell cycle arrest and activation of the p38 stress response pathway.

(A) Immunofluorescent images of RPE1 cells treated with Cep123, PCM-1, C-Nap1 or a non-targeting (NT) control siRNA, labelled with EdU (green) and stained with DAPI (blue) and an antibody against the cell proliferation marker Ki67 (red). Scale bars: 10 µm and inset 1 µm. (B) Quantification of the number of EdU-positive (S phase) and Ki67-positive (cycling) cells. The data represent the mean of three independent experiments (n = 300) with s.d. shown. Depletion of Cep123 using the Cep123 siRNA d2 caused a significant reduction in the number of both EdU- and Ki67-positive cells (student's t-test, P = 7.3×10⁻⁵ and P = 6.4×10⁻⁴, respectively), suggesting that the cells had exited the cell cycle. A significant reduction in the number of S-phase cells was also observed upon treatment with the Cep123 P1 siRNA (student's t-test, P = 2.2×10⁻²). Depletion of PCM-1, C-Nap1 and Cep123 using the Cep123 P2 siRNAs did not cause cell cycle arrest, as there was no significant change in the number of EdU- and Ki67-positive cells compared with the NT control. (C) Western blotting of lysates prepared from siRNA-treated RPE1 cells using antibodies against Cep123 (C-terminal antibody), PCM-1, C-Nap1, p38, phosphorylated p38 and α-tubulin. One set of samples was treated with 500 µM sodium arsenite for 30 minutes to induce a stress response. Regardless of the siRNA treatment used, the basal level of phosphorylated p38 was similar and treatment with sodium arsenite induced a stress response and increased levels of phosphorylated p38.