Fig. 5: Activated PKA causes enlarged Centrin signals at amplified centrosomes and phosphorylation of Centrin at serine-170.

a Quantification of interphase HCT116 and HCT-15 cells with more than two centrosomes (centrosome amplification, CA) and the proportion of centrosome amplified cells with enlarged Centrin-2 foci (Enlarged, ENL) upon activation of PKA for 48 h. Percentages of enlarged Centrin-2 foci are given. b Representative images of interphase HCT116 parental and Centrin-2 (CTN2) knockout cells co-immunostained with anti-Centrin-2 and anti-γ-tubulin antibodies (insets), and stained with Hoechst 33342. Scale bar: 10 µm. Note the absence of Centrin-2 signals in CTN2-deleted cells. c Quantification of HCT116 interphase cells (clone 1) depleted of Centrin-2 (CTN2 knockout, KO) with more than two centrosomes upon activation of GPER1 and PKA for 48 h. Whole-cell lysates were immunoblotted from cells treated with DMSO (Asyn) or 2 µM dimethylenastron (Mitotic) for 16 h (d) and (f), or were left untreated (e) and, where indicated, transfected with SCRAMBLED (SCR) or Centrin-2 specific siRNA (CETN2). α-tubulin and Vinculin served as loading controls. Relative protein levels were normalized to the loading controls and expressed relative to SCR. g Whole-cell lysates exposed or not exposed to λ-phosphatase were immunoblotted from cells treated with 2 µM dimethylenastron (mitotic). h Whole-cell lysates were immunoblotted from cells exposed to Centrin-2 phospho- and non-phosphopeptide for 2 h. Vinculin served as a loading control. i, j Cells were co-immunostained with anti-Centrin-2 and anti-Centrin-pS170 antibodies (insets), and stained with Hoechst 33342. i Representative images of cells in prometa- (PM) or anaphase (Ana) of mitosis treated as in h). Scale bar: 10 µm. j Representative images of cells in different cell cycle phases as indicated. Scale bar: 10 µm. Data information: Graphs in (a) and (c) show mean ± SD and individual data points from three different experiments with a total of 1200 (a) and 1500 (c) amplified cells. P-values < 0.05 are displayed. P-values for centrosome amplification are given in the Supplementary Table 1. The following statistics were applied: adapted bootstrap procedure for graphs in (a) and bootstrap procedure for graphs in (c) and as described in “Materials and Methods” section.