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. Author manuscript; available in PMC: 2008 Oct 28.

Published in final edited form as: Oncogene. 2008 May 19;27(40):5288–5302. doi: 10.1038/onc.2008.161

Roles of cyclins A and E in centrosome reduplication in cells arrested by exposure to aphidicolin. (A) Cyclins E^+/+ and E^−/− mouse embryonic fibroblasts (MEFs) were exposed to Aph (1 μg/ml) for 72 h. The lysates prepared from Aph-treated cells and untreated control cells were immunoblotted with anti-cyclins E and A, and anti-α-tubulin (loading control) antibodies. (B) The Aphtreated and untreated control cells were immunostained for γ-tubulin, and the number of cells with amplified centrosomes were scored. The results are shown in the graph as the average±standard error from three experiments. For each experiment,>200 cells were examined. (C) CycE^+/+/CycA RNAi and CycE^−/−/CycA RNAi cells as well as the control CycE^+/+/vec and CycE^−/−/vec cells were treated with Aph for 72 h, and the lysates prepared from Aph-treated cells and untreated control cells were immunoblotted with anticyclins E and A and anti-α-tubulin (loading control) antibodies. (D) The Aph-treated and untreated CycE^+/+/CycA RNAi, CycE^−/−/CycA RNAi, CycE^+/+/vec and CycE^−/−/vec cells were immunostained for γ-tubulin, and the number of cells with amplified centrosomes were determined. For each experiment,>200 cells were examined. The results are shown as the average±standard error from three experiments. Representative γ-tubulin immunostaining images are show in (E). In the magnified images, each centrosome is indicated by an arrow. Scale bar: 20 μm. (F)We also examined the centrosomes in the Aph-treated cells by coimmunostaining of γ- and β-tubulins after cold-treatment prior to fixation (see Materials and methods section). By this procedure, centrioles within the centrosome can be visualized. The representative immunostaining images of CycE^+/+/CycA RNAi cells after Aph treatment are shown. Scale bar: 5 μm. As described previously, centrosome amplification occurring in cells arrested by Aph is mostly due to centrosome reduplication, and not due to centrosome fragmentation (Tarapore et al., 2001). In addition, by this method, the results for the frequency of centrosome amplification were similar to those by γ-tubulin immunostaining. (G, H) CycE^−/−/CycA RNAi and CycE^−/−/vec cells were transfected with either a pEGFP-C1 vector or a plasmid encoding green fluorescent protein (GFP)-tagged cyclin E1 (see Figure 4a). The transfectants were then exposed to Aph for 72 h. The Aph-treated and untreated control cells were immunostained for γ-tubulin, and the number of cells with amplified centrosomes were scored. The results are shown in the graph as the average±standard error from three experiments (G). For each experiment, >200 cells were examined. The representative immunostaining images are shown in (H). Scale bar: 20 μm.

Roles of cyclins A and E in centrosome reduplication in cells arrested by exposure to aphidicolin. (A) Cyclins E^+/+ and E^−/− mouse embryonic fibroblasts (MEFs) were exposed to Aph (1 μg/ml) for 72 h. The lysates prepared from Aph-treated cells and untreated control cells were immunoblotted with anti-cyclins E and A, and anti-α-tubulin (loading control) antibodies. (B) The Aphtreated and untreated control cells were immunostained for γ-tubulin, and the number of cells with amplified centrosomes were scored. The results are shown in the graph as the average±standard error from three experiments. For each experiment,>200 cells were examined. (C) CycE^+/+/CycA RNAi and CycE^−/−/CycA RNAi cells as well as the control CycE^+/+/vec and CycE^−/−/vec cells were treated with Aph for 72 h, and the lysates prepared from Aph-treated cells and untreated control cells were immunoblotted with anticyclins E and A and anti-α-tubulin (loading control) antibodies. (D) The Aph-treated and untreated CycE^+/+/CycA RNAi, CycE^−/−/CycA RNAi, CycE^+/+/vec and CycE^−/−/vec cells were immunostained for γ-tubulin, and the number of cells with amplified centrosomes were determined. For each experiment,>200 cells were examined. The results are shown as the average±standard error from three experiments. Representative γ-tubulin immunostaining images are show in (E). In the magnified images, each centrosome is indicated by an arrow. Scale bar: 20 μm. (F)We also examined the centrosomes in the Aph-treated cells by coimmunostaining of γ- and β-tubulins after cold-treatment prior to fixation (see Materials and methods section). By this procedure, centrioles within the centrosome can be visualized. The representative immunostaining images of CycE^+/+/CycA RNAi cells after Aph treatment are shown. Scale bar: 5 μm. As described previously, centrosome amplification occurring in cells arrested by Aph is mostly due to centrosome reduplication, and not due to centrosome fragmentation (Tarapore et al., 2001). In addition, by this method, the results for the frequency of centrosome amplification were similar to those by γ-tubulin immunostaining. (G, H) CycE^−/−/CycA RNAi and CycE^−/−/vec cells were transfected with either a pEGFP-C1 vector or a plasmid encoding green fluorescent protein (GFP)-tagged cyclin E1 (see Figure 4a). The transfectants were then exposed to Aph for 72 h. The Aph-treated and untreated control cells were immunostained for γ-tubulin, and the number of cells with amplified centrosomes were scored. The results are shown in the graph as the average±standard error from three experiments (G). For each experiment, >200 cells were examined. The representative immunostaining images are shown in (H). Scale bar: 20 μm.