CRISPR-Mediated Kindlin-2 Knockout in E0771 and HML2 Cells

E0771 BC cells were obtained from American Type Culture Collection (ATCC) and maintained according the manufacturer’s protocols. The HML2 cells were kindly provided by Drs. Pollard and Kitamura [26 (link)]. The HML2 cell line was derived from the E0771 cells by serial propagation of the E0771-derived tumors in recipient mice [26 (link)]. Cells were also routinely authenticated by STR DNA fingerprinting analysis. Kindlin-2-KO cells were generated by lentiviral transduction using CRISPR/Cas9 gene editing as described [23 (link),24 (link),25 (link)]. We used two independent and verified Kindlin-2-specific sgRNAs for each of the human and mouse Kindlin-2 and a scrambled sgRNA (i.e., nonsilencing sgRNA, [23 (link),24 (link),25 (link)]). Loss of Kindlin-2 expression was verified by Western blot. For stimulation of cells with growth factors, cells were serum starved in serum-free DMEM growth medium without antibiotics overnight. The next day, the cells were stimulated with either 100 ng/mL EGF (Millipore) or 5 ng/mL TGF-β for 30 min. EGFR inhibitor ZD1839 and TGF-β receptor inhibitor SB431542 were obtained from SelleckChem and used at a concentration of 10 µM for 2 h. Gel electrophoresis reagents were from Bio-Rad.