Mcl 1

Mammary organoids were fixed with 4% paraformaldehyde at room temperature for 10 minutes. The organoids were washed twice with PBS, incubated with methanol for 2 minutes, followed by a 2 minutes incubation in PBS with 1% Triton-X. The organoids were permeabilized by incubating with penetration buffer (0.2% Triton-X, 0.3M glycine, 20% DMSO in PBS) for 15 minutes. Samples were then blocked with blocking buffer (5% mouse serum, 5% rabbit serum, 5% goat serum, 2% bovine serum albumin, 0.5% Triton X, 10% DMSO in PBS). MCL-1 (1:100, Santa Cruz, sc-20679) primary antibody was diluted in antibody buffer (50% blocking buffer, 0.2% Tween-20) and samples were incubated for 1 hour at room temperature on a shaker. Secondary antibody incubation with goat anti-mouse Alexa Fluor 488 (ThermoFisher A-11001) conjugated antibodies at a dilution of 1:200 was performed in the dark at room temperature for 1 hour. Samples were then washed five times before mounting on a slide using PBS:glycerol (1:1). Images were captured using a Leica SP3 confocal microscope. Images were pseudo-colored using ImageJ.

Whole cell extracts were prepared by resuspending cells in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40 substitute, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with complete protease inhibitor cocktail (Roche). Cleared lysates were boiled in Laemmli sample buffer with 1% beta-mercaptoethanol and proteins were separated by SDS-PAGE using precast 4–12% Bis-Tris gels (Life Technologies). Proteins were transferred to PVDF membrane (Millipore) at 30 V, 4 °C for 16 h. Blots were blocked for 30 min with 5% milk in TBS-T, incubated with primary antibodies for >3h and horseradish peroxidase conjugated anti-mouse IgG or anti-rabbit IgG (GE Healthcare) for 1 h, washing 4× for 10 min with excess TBS-T in between and prior to developing. Blots were developed with SuperSignal West Pico ECL substrate (Thermo Scientific) and exposed to film. The following antibodies were used in this study: Huwe1 (Bethyl, A300–486A, 1:1000), N-myc (Santa Cruz, B8.4.B, 1:500), c-Myc (Cell Signaling, 9402, 1:500), Mcl-1 (Santa Cruz, S-19, 1:200), Miz-1 (Santa Cruz, H-190, 1:200), beta catenin (Cell Signaling, 8480, 1:1000), Erk1/2 (Cell Signaling, 4695, 1:1000), phospo-Erk1/2 (Cell Signaling, D13.14.4E, 1:1000), p53 (Leica, CM5, 1:1000), vinculin (Sigma, hVIN-1, 1:5000) and actin (EMD Millipore, C4, 1:5000).