Azd7762

Gemcitabine, AZD7762, and PF477736 were purchased from Sigma (Sigma, MO, USA). Cisplatin was purchased from Tocris Bioscience (Cat#2251). Propidium Iodide Staining Solution was purchased from BD Bioscience (San Jose, CA, USA). Antibodies against CDKN1A, TP53, PARP-1, P-γH2Ax, p-Chk1-317, p-Chk-345 were purchased from Cell Signaling Tech. (New Bedford, MA). Beta-Actin antibody (Santa, Cruz) was used as protein loading control. DMEM was obtained from Cellgro (Manassas, VA, USA) and supplements were from Invitrogen (Carlsbad, CA, USA).

R26-CreERT2;R26-mTmG mice were intraperitoneally injected with 0.2 mg tamoxifen per 25 g body weight diluted in sunflower oil (Sigma) at 3 weeks of age. At 5 weeks of age, a mammary window was inserted near the fourth and fifth mammary glands (for details, see ref. 31 (link)). Mice were anaesthetized using isoflurane (1.5% isoflurane/medical air mixture) and placed in a facemask with a custom designed imaging box. Mice were intraperitoneally injected with AZD-7762 (0.5 mg in PBS, Sigma) every 5 h during the time-lapse imaging. Imaging was performed on an inverted Leica SP8 multiphoton microscope with a chameleon Vision-S (Coherent Inc.), equipped with four HyD detectors: HyD1 (< 455 nm), HyD2 (455–490 nm), HyD3 (500–550 nm) and HyD4 (560–650 nm). Different wavelengths between 700 nm and 1,150 nm were used for excitation; collagen (second harmonic generation) was excited with a wavelength of 860 nm and detected in HyD1. GFP and Tomato were excited with a wavelength of 960 nm and detected in HyD3 and HyD4. TEBs and ducts were imaged every 20–30 min using a Z-step size of 3 μm over a minimum period of 8 h. All images were in 12 bit and acquired with a 25× (HCX IRAPO N.A. 0.95 WD 2.5 mm) water objective.

GEM and AZD7762 were purchased from Sigma-Aldrich (St. Louis, MO, USA)
and Selleckchem (Houston, TX, USA), respectively. TL32711 was provided by
TetraLogic Pharmaceuticals (Malvern, PA, USA). Human breast cancer cell lines
HCC1806, HCC38, MB468, BT549, Hs678T, MB231, HCC70, HCC1937, HCC1395, and BT20
were purchased from the American Type Culture Collection (Manassas, VA, USA).
All cell lines were grown in RPMI medium 1640 supplemented with 10% (v/v) fetal
bovine serum and 100 mM penicillin-streptomycin at 37°C, which were
purchased from Invitrogen (Carlsbad, CA, USA). TNF-α neutralizing
antibody and caspase inhibitor z-VAD-FMK were purchased from Cell Signaling
(Danvers, MA, USA) and Promega (Madison, WI, USA), respectively.