Cell proliferation and apoptosis are assessed by staining for a proliferation marker, Ki67, or an apoptotic marker, cleaved caspase 3, following the protocol described above for the polarity markers. MCF10 variants are grown in 3D rBM overlay cultures for 4 days and are treated with 0.1 % DMSO, 5 μM CA074Me or 5 μM Aloxistatin. The percentage of structures that are positive for Ki67 or cleaved caspase 3 is determined by counting a total of 100 structures on two separate coverslips with a Zeiss Axiophot epifluorescent microscope. Structures are considered Ki67 positive if they contained at least one cell staining for Ki67. Structures are considered to be caspase 3 positive if they contained at least one cell that is positive for cleaved caspase 3 and the positive cell(s) is not localized in the center of a developing lumen^[3].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice and Pigs^[4]
Guinea Pigs (male, Hartley strain, average weight 400 g corresponding to animals about 6 weeks old) are used. Male transgenic mice expressing human AβPP containing the wt β-secretase site and the London mutant β-secretase site sequences are used. Delivering a drug by gavage offers the advantage of accurate dosing but is traumatic and thus only suitable for relatively short dosing periods (up to about a week). Delivery by gavage is used for the guinea pig studies. Aloxistatin is suspended in Me2SO at the indicated concentrations (0.1, 1.0, 5, and 10 mg/kg) and administered by gavage daily using a feeding tube. Vehicle control animals are treated by gavage of Me2SO alone.
Rats^[5]
Male inbred DS rats are used. Weaned rats are fed laboratory chow containing 0.3% NaCl until 7 weeks of age. DS rats fed an 8% NaCl diet after 7 weeks manifest compensated concentric left ventricular (LV) hypertrophy secondary to hypertension at 12 weeks and a distinct stage of fatal LV failure with lung congestion at 19 weeks. DS rats are therefore fed an 8% NaCl diet from 7 weeks of age and are randomized to an HF group, an Aloxistatin group (10 mg per kg of body mass per day, administered intraperitoneally every other day), or an RNH-6270 group (3 mg/kg per day in chow) from 12 to 19 weeks of age (n=10 for each group). The doses of RNH-6270 (an ARB) and Aloxistatin are determined in preliminary experiments and previous studies. DS rats maintained on the 0.3% NaCl diet served as age-matched controls (control group, n=10). At 19 weeks of age, all of the rats are euthanized by an intraperitoneal overdose of NSC 10816 (50 mg/kg), and the hearts are removed for biological and histological analyses. Arterial blood is collected from the abdominal aorta for the measurement of renin activity. Systolic blood pressure and heart rate are measured in conscious rats from 7 weeks of age, every week, using a noninvasive tail-cuff method. In separate experiments, 12-week-old DS rats, fed a low-salt diet from 7 weeks of age, are given vehicle, RNH-6270, or Aloxistatin in the same manner as in the above experiments (n=5 for each group), and the LV tissues for measuring targeting mRNAs and protein levels are immediately placed in liquid nitrogen and stored at -80°C.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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