Figure 5. Cells lacking Ki-67 enter the cell cycle efficiently.
(A) Top, re-entry of cell cycle in NIH-3T3 WT clone W4 and Ki-67-negative mutant clones 60 and 65 after serum starvation-induced cell cycle arrest. Progression of cell cycle entry analysed by FACS using EdU staining. Bottom, quantification of cell cycle phases in this experiment. (B) Western blot analysis of Ki-67 (upper panel) and cyclin A2 (lower panel) upon cell cycle entry. LC, loading control.
Figure 5—figure supplement 1. Cells lacking Ki-67 proliferate efficiently.
(A) Schematic presentation of generation of Ki-67 shRNA knockdown BJ-hTERT. (B) Left, Western blot for indicated proteins upon cell cycle re-entry in serum starved hTERT-transformed BJ fibroblasts (BJ-hTERT) after induction of shRNA against Ki-67 (+) or GAPDH control (-). LC, loading control. Right, DNA synthesis analysed by flow cytometry after EdU pulse. (C) Left, asynchronous BJ-hTERT with doxycyclin-induced control or Ki-67 shRNA-expression were additionally transfected with control or Ki-67 siRNA for 48 hr. Protein levels were analysed by Western blotting. LC, loading control. Right, cell cycle distribution by flow cytometry.
Figure 5—figure supplement 2. Cells lacking Ki-67 proliferate efficiently.
(A) Schematic presentation of generation of Ki-67 shRNA knockdown cell lines. (B) Top: Western blot analysis of the indicated proteins in asynchronously growing U2OS and HeLa cells stably expressing non-targeting (CTRL) or Ki-67 shRNA. Lanes separated by lines were from a single exposure of a single SDS-PAGE gel and Western blot. LC, loading control. Middle: Cell cycle distribution of these cells. Bottom: qRT-PCR analysis of the indicated mRNA levels, normalized by mRNA expression of beta-2-microglobulin (B2m). (C) Immunofluorescence of Ki-67 and EdU in asynchronous U2OS cells stably expressing control or Ki-67 shRNA, incubated with 5-ethynyl-2’-deoxyuridine (EdU) for 3 hr. Bar, 10 µm. Right: Ratio of EdU-positive cells to the total cell number for each incubation time; ns: not significant.