Zd1839

The cultured cells at 60% confluence were incubated with 1% FBS-containing media for 24 h followed by treatment of human TNC (Millipore, CC605, 5 μg/mL or 10 μg/mL) or TGFβ (2 ng/mL to 10 ng/mL, Sigma, APREST95171). The STAT3 inhibitor Stattic (Selleck, S7024, 2 μM) or EGFR inhibitor Gefitinib (5 μM, Selleck, ZD-1839) were added to the media 30 min before TNC treatment.

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.

miPS-CSCs were seeded with a density of 1 × 10⁴ cells/well on 96-well plates coated with gelatin. The concentrations of Gefitinib (Selleck, ZD1839) and Eganelisib (Selleck, S8330) were formulated according to the manufacturer’s instructions. After 24 h, test compounds were added at a concentration gradient of 3.125 μmol/L, 6.25 μmol/L, 12.5 μmol/L, 25 μmol/L and 50 μmol/L. Cell viabilities at 48 h were determined as follows. After incubation, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, yellow tetrazole (MTT, Sigma-Aldrich) were added to the wells with the final concentration of 0.6 mg/mL and the plate was incubated for 4 h. Formed formazan crystals were dissolved in 10% (w/v) SDS with 0.02 N HCl and incubated overnight. Finally, the absorbance of each well was measured at 570 nm using an MTP-800AFC microplate reader (Corona, Japan). Viable cells were evaluated by MTT assay as described above. The half-maximal inhibitory concentration (IC50) values were determined based on the survival curve obtained by MTT assay. The experiment was independently repeated three times.