Foxc1

The total protein content from the cells was extracted in cell RIPA buffer containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate, supplemented with a mixture of complete protease inhibitors and phosphatase inhibitors. After centrifugation, the protein in the supernatant was collected and stored at −20 °C. The protein samples were determined using the Lowry protein assay. Briefly, equal amounts (20–30 μg) of protein were heated after adding the appropriate amount of 5× sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (40% glycerol, 10% SDS, 5% 2-mercaptoethanol, 0.02% bromophenol blue, 250 mM Tris–HCl, pH 8.8). The samples were separated on 6%–15% SDS-PAGE and subsequently transferred onto nitrocellulose filters using the Bio-Rad electrotransfer system (Bio-Rad Laboratories, Munich, Germany). The blots were then incubated with specific primary antibodies (1:1000) overnight at 4 °C, washed three times with TBST, and incubated with the appropriate secondary antibody for 1 h at room temperature. β-Actin, LATS2, Ki-67, α-SMA (Abcam, Cambridge, United Kingdom), ERK1 (BD Biosciences), p-Akt, p-ERK1/2, Akt, p21, p27, α-tubulin, HOAC1, and FOXC1 (Santa Cruz Biotechnology) primary antibodies were used. Finally, the blots were developed using a custom-made ECL detection system.

The #4 mammary glands from wildtype control mice and MMTV-FOXC1 transgenic mice were fixed in formalin (10%) and paraffin-embedded by the Cedars-Sinai Medical Center pathology lab. Sectioning and H&E staining were also performed. Immunohistochemistry was conducted using Vectastain ABC Kit (Vector laboratories) and ImmPACT DAB Kit (Vector). Sections were deparaffinized and rehydrated by xylene and gradient ethanol, respectively. An antigen retrieval method using microwave pretreatment and 0.01 M sodium citrate buffer (pH6) was used for all antibodies (Vector laboratories, Burlingame, CA). Primary antibodies were FOXC1 (1:100, sc-21394, Santa Cruz), Myc-Tag (1:100, #2272, Cell Signaling), ER (1:100, sc-542 Santa Cruz), PR (1:100, sc-538 Santa Cruz), Ki67 (1:100, 790–4286, Roche Diagnostics), Elf5 (1:100, sc-9645, Santa Cruz), Keratin 5 (1:100, 905501, Biolegend), Keratin 8 (1;100, DSHB), p-Stat5(1:100,9359 s, Cell Signaling). The signal was visualized using the VECTASTAIN ABC Systems (Vector laboratories). Counterstaining was performed with Mayer’s hematoxylin (Sigma). The percentages were calculated by comparing stained nuclei to total nuclei in epithelial cells.

Proteins were extracted from mouse mammary tissue which was homogenized with RIPA lysis buffer (Sigma-Aldrich) and then centrifuged 20 min twice at 13,000 rpm. Protein concentration was determined by the BCA Protein Assay Kit (ThermoFisher, Waltham, MA). Proteins (50 ug) were run on 4–20% gradient gels and transferred onto PVDF membranes using a Trans-Blot Turbo transfer pack and Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with Odyssey blocking buffer (LI-COR) and incubated with primary antibodies overnight at 4 °C. The primary antibodies were FOXC1 (1:500, sc-21394, Santa Cruz), Actin (1:500, sc-1616, Santa Cruz), Elf5 (1:200, sc-9645, Santa Cruz), and Lamin A/C (1:500, sc-7292, Santa Cruz), p-Stat5 (1:200, 4322 s, Cell signaling), Stat5(A-9) (1:200, sc-74442, Santa Cruz).