Figure 9.
Figure 9. Golgi localization is required for RhoBTB3 regulation of Cyclin E levels, Golgi structure, and cell size. (A) HeLa cells were treated with Control (top) or RhoBTB3 siRNA (as indicated) for 72 h. After 24 h, cells were transfected with plasmid encoding siRNA-resistant Myc-tagged WT-RhoBTB3 or Myc-tagged 1–525 truncation. Left column, Cyclin E1 detected with mouse anti–Cyclin E (HE12). Middle (in top and second panels), nuclei stained with DAPI; right column, TRITC-labeled RhoBTB3 siRNA #1. Red asterisks indicate siRNA-transfected cells, yellow asterisks indicate cells transfected with siRNA and rescue plasmid. Bars, 40 µm. (B and C) Quantification of rescue. Control siRNA (black column), RhoBTB3 siRNA (red column), RhoBTB3 siRNA + WT rescue construct (blue column), and RhoBTB3 siRNA + 1–525 rescue construct (green column). The percentages of Cyclin E–positive cells (B) and nuclear Cyclin E intensity (C) were obtained using an unbiased quantification method (see Materials and methods). Data represent the mean of three independent experiments in which a total of >1,000 cells were analyzed in control and RhoBTB3 siRNA conditions, or ≥398 cells in rescue conditions. t test; *, P < 0.05; **, P < 0.01; error bars represent SEM. (D and E) Interaction of WT-RhoBTB3 or 1–525 RhoBTB3 construct with FLAG–Cyclin E (D) and FLAG-CUL3 (E) in total lysates from HEK293T cells. Cells were transfected as indicated at the top, immunoprecipitated with anti-FLAG antibody, and bound proteins were detected by immunoblot with antibodies to the proteins indicated at the right. Molecular mass marker mobility is shown at the left in kilodaltons. (F) HeLa cells were treated with indicated siRNA for a total of 72 h. At 24 h after siRNA, cells were transfected with plasmid encoding siRNA-resistant Myc-tagged WT-RhoBTB3 (top) or Myc-tagged 1–525 truncation (bottom). Left, Golgi structure assessed using mouse anti-GCC185. Middle, RhoBTB3 rescue construct expression detected using chicken anti-Myc. Right, TRITC-labeled siRNA. Asterisks are as in A above. Bars, 40 µm. (G and H) Quantification of Golgi fragmentation (G) and cell area (H). Color code as in B. Data in G and H represent the mean of four independent datasets in which a total of 150–200 cells were analyzed in all conditions. t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; error bars represent SEM.

Golgi localization is required for RhoBTB3 regulation of Cyclin E levels, Golgi structure, and cell size. (A) HeLa cells were treated with Control (top) or RhoBTB3 siRNA (as indicated) for 72 h. After 24 h, cells were transfected with plasmid encoding siRNA-resistant Myc-tagged WT-RhoBTB3 or Myc-tagged 1–525 truncation. Left column, Cyclin E1 detected with mouse anti–Cyclin E (HE12). Middle (in top and second panels), nuclei stained with DAPI; right column, TRITC-labeled RhoBTB3 siRNA #1. Red asterisks indicate siRNA-transfected cells, yellow asterisks indicate cells transfected with siRNA and rescue plasmid. Bars, 40 µm. (B and C) Quantification of rescue. Control siRNA (black column), RhoBTB3 siRNA (red column), RhoBTB3 siRNA + WT rescue construct (blue column), and RhoBTB3 siRNA + 1–525 rescue construct (green column). The percentages of Cyclin E–positive cells (B) and nuclear Cyclin E intensity (C) were obtained using an unbiased quantification method (see Materials and methods). Data represent the mean of three independent experiments in which a total of >1,000 cells were analyzed in control and RhoBTB3 siRNA conditions, or ≥398 cells in rescue conditions. t test; *, P < 0.05; **, P < 0.01; error bars represent SEM. (D and E) Interaction of WT-RhoBTB3 or 1–525 RhoBTB3 construct with FLAG–Cyclin E (D) and FLAG-CUL3 (E) in total lysates from HEK293T cells. Cells were transfected as indicated at the top, immunoprecipitated with anti-FLAG antibody, and bound proteins were detected by immunoblot with antibodies to the proteins indicated at the right. Molecular mass marker mobility is shown at the left in kilodaltons. (F) HeLa cells were treated with indicated siRNA for a total of 72 h. At 24 h after siRNA, cells were transfected with plasmid encoding siRNA-resistant Myc-tagged WT-RhoBTB3 (top) or Myc-tagged 1–525 truncation (bottom). Left, Golgi structure assessed using mouse anti-GCC185. Middle, RhoBTB3 rescue construct expression detected using chicken anti-Myc. Right, TRITC-labeled siRNA. Asterisks are as in A above. Bars, 40 µm. (G and H) Quantification of Golgi fragmentation (G) and cell area (H). Color code as in B. Data in G and H represent the mean of four independent datasets in which a total of 150–200 cells were analyzed in all conditions. t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; error bars represent SEM.

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