MDA-MB-231 and MDA-MB-468 and HME cells were purchased from the American Type Culture Collection and grown under recommended conditions. Cell lines were authenticated using STR profiling, and were tested for mycoplasma infection using Sigma LookOut Mycoplasma PCR Detection Kit (Cat MP0035). CCN6 shRNA and scrambled controls were reported previously [18 (link)]. The scrambled control vector shRNA-SC and IGF2BP2 knockdown shRNA plasmid (pLKO.1-puro was purchased from Sigma (St. Louis, MO) and packaged at the University of Michigan Vector Core. For stable transductions, the virus-containing supernatant was diluted 1:1 with fresh media and supplemented with 8 μg/ml polybrene to infect HME cells. Selection was initiated in 10 μg/ml puromycin (Sigma-Aldrich Co., St. Louis, MD) 48 hours after infection. Recombinant CCN6 protein (rCCN6) (200 ng/mL) was purchased from PeproTech, and used as in our previous studies [18 (link)].
For MDA-MB-231 and −468 breast cancer cells and for HME non tumorigenic breast cells, Western blot analyses were carried out with 100μg of whole cell extract derived as previously reported [19 (link)]. Primary antibodies used include: CCN6, IGF2BP2, HMAG2 (Abcam, #ab187666, #ab129071, #ab97276, respectively), E-cadherin, ZEB1, SNAI2/Slug, (Cell Signaling, #3195, #3396, #9585 respectively); β-Actin (Santa Cruz Biotechnology, #SC-47778).
For MDA-MB-231 and −468 breast cancer cells and for HME non tumorigenic breast cells, Western blot analyses were carried out with 100μg of whole cell extract derived as previously reported [19 (link)]. Primary antibodies used include: CCN6, IGF2BP2, HMAG2 (Abcam, #ab187666, #ab129071, #ab97276, respectively), E-cadherin, ZEB1, SNAI2/Slug, (Cell Signaling, #3195, #3396, #9585 respectively); β-Actin (Santa Cruz Biotechnology, #SC-47778).
