Fig. 2

CEP85 is required for PLK4 activation and STIL localization to centrosomes. a–c Examining the impact of CEP85 depletion on PLK4 activation using the PLK4-induced centriole overduplication assays (see Methods). Selected images showing Myc-PLK4 and PLK4 pS305 labelling. Scale bar 10 μm, white boxes indicate the magnified region. b, c The graph indicates the levels of Myc-PLK4 and the relative ratio of pS305/PLK4 at centrosomes after depletion of endogenous CEP85. (n = 200/experiment, three independent experiments). d–f Western blot showing the levels of Myc-PLK4, CEP85 and STIL in control or CEP85 siRNA transfected cells. Cyclin A was used as a cell cycle marker and α-tubulin served as a loading control. e, f Quantification of protein levels shown in D (α-tubulin normalized, n = 2/experiment, five independent experiments,). g, h IF analysis of STIL localization in control or CEP85-depleted cells. The S-phase arrest assays were performed as described in Methods, followed by labelling with DAPI and the indicated antibodies. Scale bar 10 μm, white boxes indicate the magnified region. h Quantification showing the relative levels of STIL at centrosomes in PCNA-positive cells (n = 200/experiment, three independent experiments). i, j Western blot indicates the levels of STIL and CEP85 in control or CEP85 siRNA treated cells. Cyclin A and α-tubulin served as a cell cycle marker and loading control, respectively. j Quantification of the indicated protein levels shown in I (α-tubulin normalized, n = 2/experiment, five independent experiments). Two-tailed t-test was performed for all p-values, all error bars represent SD, and asterisks for p-values are **p < 0.01 and *p < 0.05