Figure 5To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing (endogenous) γ-tubulin-EGFP (A) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of γ-tubulin (A) molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: γ-tubulin: 45; in late S/G2 phase: γ-tubulin: 45; in late G2 phase: γ-tubulin: 48; in early mitosis: γ-tubulin: 31. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05). To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing Sas-6 (B) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: Sas-6: 43; in late S/G2 phase: Sas-6: 46; in late G2 phase: Sas-6: 48. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05). To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing STIL (C) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of STIL (C) molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: STIL: 79; in late S/G2 phase: STIL: 68; in late G2 phase: STIL: 68. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).