Figure 4. CDKN3 is a nucleolar protein... | Rockefeller University Press

Figure 4.

$Figure 4. CDKN3 is a nucleolar protein during interphase and controls G1/S transition. (A) Endogenous CDKN3 localizes to interphase nuclei. Detergent extraction before fixation helps visualize the nucleolar fraction of endogenous CDKN3 (bottom). The signal is lost from cells transfected with CDKN3 siRNA (right), demonstrating antibody specificity. (B) Endogenous CDKN3 colocalizes with nucleolin. CDKN3 foci (green) are surrounded by diffuse nucleolin staining (red) in each nucleolus. (C) CDKN3 phosphatase activity is not required for subcellular targeting of GFP-CDKN3. HeLa cells were transfected with wild-type GFP-CDKN3 (top) and phosphatase-dead GFP-CDKN3C79S point mutant (bottom). Nucleolar GFP-CDKN3 is visible in detergent-extracted nuclei. At least 100 nuclei were visualized per construct and condition. (D) Representative cell cycle profiles of cells transfected with control siRNA and CDKN3 siRNA. (E) Mildly increased S-phase fraction in HeLa cells transfected with CDKN3 siRNA. P < 0.01 in t test; n = 4. (F) CDKN3 is required for serum starvation–induced G1 arrest. Serum-starved HCT cells transfected with control and CDKN3 siRNAs were pulsed with EdU in serum-free medium to visualize cells that enter S phase (see micrographs on the right; red arrows indicate EdU-positive nuclei). P < 0.0001 in t test; n = 4.$

CDKN3 is a nucleolar protein during interphase and controls G1/S transition. (A) Endogenous CDKN3 localizes to interphase nuclei. Detergent extraction before fixation helps visualize the nucleolar fraction of endogenous CDKN3 (bottom). The signal is lost from cells transfected with CDKN3 siRNA (right), demonstrating antibody specificity. (B) Endogenous CDKN3 colocalizes with nucleolin. CDKN3 foci (green) are surrounded by diffuse nucleolin staining (red) in each nucleolus. (C) CDKN3 phosphatase activity is not required for subcellular targeting of GFP-CDKN3. HeLa cells were transfected with wild-type GFP-CDKN3 (top) and phosphatase-dead GFP-CDKN3^C79S point mutant (bottom). Nucleolar GFP-CDKN3 is visible in detergent-extracted nuclei. At least 100 nuclei were visualized per construct and condition. (D) Representative cell cycle profiles of cells transfected with control siRNA and CDKN3 siRNA. (E) Mildly increased S-phase fraction in HeLa cells transfected with CDKN3 siRNA. P < 0.01 in t test; n = 4. (F) CDKN3 is required for serum starvation–induced G1 arrest. Serum-starved HCT cells transfected with control and CDKN3 siRNAs were pulsed with EdU in serum-free medium to visualize cells that enter S phase (see micrographs on the right; red arrows indicate EdU-positive nuclei). P < 0.0001 in t test; n = 4.