Ab75895

BT474 was cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic under a 5% CO2 environment. BT474 PTEN- LTT cells were generated by lentiviral infection to introduce PTEN shRNA, followed by single cell colony formation to establish a genetically identical cell line. Long term treatment with trastuzumab was carried out for greater than 3 months as previous described [10 (link), 49 (link)], and were otherwise maintained in the same media as the parental cell line. Sulforaphane was obtained from LKT Laboratories. Polyclonal antibodies against MEOX1 were purchased from Abcam (ab75895).

Western blotting was performed as described previously (Li et al., 2016 (link)). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. Protein bands were visualized using a chemiluminescent detection system (Thermo Scientific), and images were captured with a scanner. Antibodies against Meox1 (ab75895; Abcam) and GAPDH (ab181602; Abcam) were used for Western blotting.

Chromatin immunoprecipitation (ChIP) was performed using a ChIP assay kit (Beyotime, Shanghai, China) and Dynabeads Protein G (Invitrogen) as described previously (Mehra and Wells, 2015 (link)). The qPCR primer sequences used are shown in Supplementary Table S5. Antibodies against Smad2 (ab71109; Abcam, Cambridge, England), Smad3 (ab28379; Abcam), Smad4 (AF2097, R&D), Meox1 (ab75895; Abcam), polymerase II (ab5095; Abcam), and immunoglobulin G (IgG) (ab2410; Abcam) were used for ChIP assays.