Figure 7.

DNA damage-induced cell cycle arrest and senescence in CNOT1-depleted HeLa cells. (A,B) Up- and downregulated genes 72 h post CNOT1 siRNA treatment in HeLa cells. (C–E) Representative immunoblots showing the comparative protein expression between control and CNOT1-depleted HeLa cells. GAPDH and CDK2 were used as loading controls. (F,G) HeLa cells were transfected with either Control or CNOT1 siRNA and stained with PI and the cell cycle profiles analysed by flow cytometry at different time-points post-transfection. Representative profiles of the cells 72 h post transfection are shown. (H) HeLa cells were plated at appropriate concentrations 72 h post CNOT1 and control siRNA transfection. At day 14, large colonies were stained with crystal violet and counted. (I) Bar graph shows the plating efficiency of the cells at day 14 after staining with crystal violet (n = 3 independent experiments). Statistical analyses were performed using a two-tailed and unpaired Student’s t test, ***, p < 0.001. Error bars represent SD. (J) Representative images showing the development of SA-β-Gal staining (positive senescent cells) in control HeLa cells (upper panel) and CNOT1-depleted HeLa cells (lower panel) between day 3 (left panel) and day 7 (right panel) post siRNA transfection. The blue color represents senescent cells. Scale bar = 50 μm. (K) Bar graphs showing the percentage of SA-β-Gal positive cells in control and CNOT1-depleted HeLa cells 3-, 5- and 7-days post siRNA depletion (n = 3 independent experiments). Statistical analyses were performed using a two-tailed and unpaired Student’s t test, * p < 0.05. Error bars represent SD.