Figures & data
Figure 1. Y-29794 inhibits endopeptidase activity in TNBC cell lysates
(a) PREP levels were determined by immunoblotting in the indicated TNBC cell lines. Actin was used as a loading control. (B) Endopeptidase activity in lysates from the indicated TNBC cells was determined in the presence of increasing Y-29794. (c) Endopeptidase activity was determined in MDA231 and SUM159PT cells infected with control lentivirus (Csh) or lentivirus expressing shRNA against PREP (PREPsh).
Figure 2. Y-29794 inhibits proliferation and viability of triple-negative breast cancer cell lines
The indicated triple-negative breast cancer cell lines plated in a 96-well plate. One day later (D1) the cells were untreated or treated with increasing doses of Y-29794, and MTT absorbance was determined 4 days later (D4). MTT absorbance increased between D1 and D4 in untreated cells, indicating the cells were proliferating. The average results from three experiments are plotted.
Figure 3. Y-29794 inhibits viability of triple-negative breast cancer cells
The indicated TNBC cell lines were plated in 60 mm dishes and 1 day after plating were either untreated or treated with 10 µM Y-29794 for increasing amounts of time (12–48 h). The cells were then rinsed twice with PBS, refed with drug-free medium (minus Y-29794), and allowed to grow for 5 days. The cells remaining on the plate were then stained with crystal violet.
Figure 4. Y-29794 affects the cell cycle in TNBC cells
MDA468 and MDA231 cells were treated with increasing doses of Y-29794 for 48 h and the percentage of cells in each cell cycle phase was determined by flow cytometry. The tables on the right indicate the raw data, which is graphed on the left.
Figure 5. Y-29794 inhibits the IRS1/AKT/mTORC1 pathway in triple-negative breast cancer cells
(a) The indicated TNBC cell lines were treated for 16 h with increasing doses of Y-29794 (5, 7.5 or 10 µM) and examined by immunoblotting for the indicated proteins. (b) MDA468 cells were treated with 5 or 7.5 µM Y-29794 alone or in combination with proteasome inhibitor MG132 (10 µM) for 8 h. IRS1 protein levels were determined by immunoblotting. Actin served as loading control.
Figure 6. Inhibitors of IRS1, AKT, and mTORC1 reduce viability in TNBC cells
(a) MDA231 cells were plated in 35 mm dishes and treated 1 day after plating with NT157 (2.5 µM), MK2206 (10 µM), or rapamycin (10 µM). Treatment continued for 72 h, and cells remaining on the plates were then stained with crystal violet. (b) MDA231 cells were treated with MK2206 (10 µM) for 72 h or NT157 (10 µM) or rapamycin (10 µM for 24 h and examined by immunoblotting for the indicated proteins. Arrows indicate the p70 and p85 phosphorylated S6K bands. (c) MDA468 cells were plated in 35 mm dishes and treated 1 day after plating with NT157 (10 µM), MK2206 (10 µM), or rapamycin (10 µM). Treatment continued for 72 h, and cells remaining on the plates were then stained with crystal violet. (d) MDA468 cells were treated with NT157 as indicated, MK2206 (10 µM), or rapamycin (10 µM) for 72 h and examined by immunoblotting for the indicated proteins. Arrows indicate the p70 and p85 phosphorylated S6K bands.
Figure 7. The effect of PREP knockdown on the IRS1/AKT/mTORC1 pathway and proliferation in triple-negative breast cancer cells
(a) MDA231 and SUM159PT were isolated after infection with control lentivirus or lentivirus encoding shRNA against PREP. Immunoblotting for PREP and the indicated proteins in the IRS1/2/AKT/mTORC1 pathway is shown. (b) PREP mRNA levels in the control and shPREP cells were determined by qRT-PCR. (c) 100,000 shCtrl and shPREP MDA231 and SUM159PT cells were plated in multiple 35 mm dishes. Cell counts were obtained 2 and 4 days after plating. The average results from two experiments are plotted.
Figure 8. Y-29794 inhibits TNBC xenograft tumor growth
1x106 MDA468, SUM159PT, and MDA231 cells were seeded into the mammary fat pad of NOD-SCID immunocompromised mice for tumor growth. When tumors reached a palpable, measurable size, the mice were treated daily (5 days/week) with either vehicle (saline for MDA468 and SUM159PT, cremaphor for MDA231) or two doses of Y-29794, as indicated. Treatment was by intraperitoneal injection. Tumor growth was monitored by caliper measure for 3–5 weeks, and overall body weight measured as an indicator of toxicity. Plotted are tumor volumes (left) and % body weight (right). MDA468 and SUM159PT had an n = 8 for all groups (vehicle and Y-29794 treated). For MDA231 had an n = 3 for the control group, and an n = 4 for the Y-29794 treatment groups.
