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Supplementary Figure 1. Treatment with MLN8237 induces markers of cellular senescence in two
additional CAL-51 p53 and p73 shRNA knockdown clones (p53-12, p73-26). This figure supplements
the data shown in Figure 3 of the main manuscript. CAL-51 p53-12 and p73-26 knockdown clones were
treated with vehicle or MLN8237 (100 nmol/L) for the indicated time points and then stained for
senescence-associated β-galactosidase activity (blue stain) as described in Materials and Methods.
Representative images were taken at 20X magnification. Scale= 10 µm.
Supplementary Figure 2. Quantification of total cell counts and average cellular size at Day 15 as
depicted in Fig. 4A. Images from three different representative fields of view were taken at each time
point. The number of cells in each field was counted and an average with standard deviation calculated.
The average cellular size was obtained by selecting a representative total of ten cells from each of the
three fields of view followed by calculating the average with standard deviation. Please see
Supplementary Table X for additional data.
Supplementary Figure 3. Effects of MLN8237 on markers of apoptosis and senescence. A-B)
Immunofluorescence analysis of p53 wildtype CAL-51 TNBC cell line stably transfected with a p73
shRNA construct (p73-55). Cells were treated with vehicle or MLN8237 at 50 nmol/L and 100 nmol/L
for 4 days and stained with the indicated antibodies as described in Materials and Methods. C-D)
Immunofluorescence analysis of CAL-51 cell line stably transfected with either a p53, p73, or empty
SCR shRNA construct. Cells were treated with vehicle or MLN8237 at 50 nmol/L and/or 100 nmol/L for
4 days and stained with the indicated antibodies as described in Materials and Methods. Representative
images were acquired using confocal microscopy at 60X magnification. Scale= 10 µm.
Supplementary Figure 4. Effect of MLN8237 on Cleaved Caspase-3 in TNBC xenografts in vivo.
Histologic analysis of tumors from A) CAL-51, B) CU_TNBC_003, and C) CU_TNBC_002 animal
models depicted in Figure 6 A, D and F. Tumors were harvested at denoted time points, formalin fixed,
paraffin embedded and 5 micron sections were stained for Cleaved Caspase-3 and hematoxylin/eosin
(H&E). Representative images of Cleaved Caspase-3, and H&E staining were taken at 10X
magnification. Scale= 10 µm.