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. 2017 May 24;8(29):47916–47930. doi: 10.18632/oncotarget.18147

Figure 1. MDM2 depletion in estrogen-treated breast cancer cells inhibits colony formation in 3D culture conditions.

Figure 1

(A) Number of large colonies (50μm or larger) determined by counting the colonies of MCF7 cells when grown in soft agar in the presence of estrogen and in the presence or absence of shRNA induction (viewed by inverted fluorescence microscope). Average of three independent experiments are shown. The number of colonies for 3 independent experiments were as follows: control vector –shRNA induction (160, 158, 175), control vector + shRNA induction (153, 151, 197), mdm2shRNA –shRNA induction (140, 137, 200) and mdm2shRNA + shRNA induction (17, 11, 8). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.002. (B) Number of large colonies (100μm or larger) determined by counting the colonies of T47D cells when grown in soft agar in the presence of estrogen and in the presence or absence of shRNA (viewed by inverted fluorescence microscope). Average of three independent experiments are shown. The number of colonies for 3 independent experiments were as follows: control vector –shRNA induction (220, 191, 70), control vector + shRNA induction (202, 195, 33), mdm2shRNA –shRNA induction (166, 175, 69) and mdm2shRNA + shRNA induction (27,20, 9). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.02. (C) Representative images of colonies that T47D cells formed in soft agar in the presence or absence of MDM2 knockdown. (D) T47D cells grown in matrigel for 3 weeks in presence of estrogen and in presence or absence of 4μg/ml doxycycline, were fixed and stained with propidium iodide. Colony masses were categorized in 5 different groups and the number of masses in each group were counted and presented as percentages in the total population. This is an average of two independent experiments. The total number of masses scored for 2 independent experiments were control vector –shRNA induction (20, 15), control vector + shRNA induction (25, 27), mdm2shRNA –shRNA induction (93, 95) and mdm2shRNA + shRNA induction (86, 83). The p-value was determined by 2-tailed Student t-test. The p-value for large and small colonies for comparisons with and without MDM2 knockdown were p-value=0.03 and p-value=0.05 respectively. Two independent scorers counted the numbers of colonies for each independent experiment.