Gel electrophoresis reagents

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.

We used the following antibodies: rabbit anti pAKT-S473, pAKT-T308, AKT, pERK1/2, ERK1/2, WAVE3 (Cell Signaling Technology); rabbit anti pSMAD3 and SMAD3 (Abcam); mouse anti-GFP (Clontech) (1:1000); goat horseradish-peroxidase-conjugated anti-mouse IgG and goat horseradish-peroxidase-conjugated anti-rabbit IgG (Calbiochem) (1:2000); mouse monoclonal anti-Actin (Sigma) (1:5000); and Alexa-Fluor-Plus-594-conjugated anti-rabbit IgG and Alexa-Fluor-Plus-488-conjugated anti-mouse IgG (Invitrogen). Vecta-shield with DAPI was from Vector Laboratories. Gel electrophoresis reagents were from Bio-Rad.

TNBC Cell lines were acquired from the American Type Culture Collection (ATCC; Manassas, VA), and maintained according to the manufacturer’s established protocols. Cell lines were also routinely authenticated by short tandem repeat DNA fingerprinting analysis. WAVE3-deficient (W3-KO) cells were generated using two different and verified sgRNAs targeting the human WAVE3 and the mouse Wave3 genes by lentiviral transduction as described previously [11 (link)]. A scrambled (SCRAM) sgRNA was used as negative control [11 (link)]. Generation of the WAVE3-KO cells overexpressing wildtype (WT) or phosphomutant (Y4) were described previously [17 (link)]. GFP-tagged WAVE3 constructs were generated as described previously [17 (link)]. The GFP-recombinant vector or the empty GFP expression control vector was used for stable transfections using standard protocols. We used the following reagents: Doxorubicin, docetaxel and cisplatin, purchased from Sigma (St. Louis, MO) as powders, were dissolved in DMSO and used as described previously [12 (link)]. For proteasome inhibition, cells were treated with GM6001 (Sigma) at 20 µM for 24 h. Gel electrophoresis reagents were from Bio-Rad (Hercules, CA).

The following primary antibodies were from Cell Signaling Technology: Rabbit anti α-Tubilin (1:1000), mouse anti-p53 (1:1000) and rabbit anti-p53 (1:1000 for WB and 1:500 for immunofluorescence). Mouse monoclonal anti-Kindlin-2, clone 3A3 (1:2000 for WB and 1:500 for immunofluorescence) was from Millipore; rabbit polyclonal anti SerpinB2 (1:1000) and rabbit polyclonal anti Lamin B1 (1:3000) were from Abcam. Goat horseradish peroxidase-conjugated anti-mouse IgG (1:2000) and goat horseradish peroxidase-conjugated anti-rabbit IgG (1:2000) were from Calbiochem. Mouse monoclonal anti-Actin (1:5000) was from Sigma. Alexa Fluor Plus 594-conjugated anti-Rabbit IgG and Alexa Fluor Plus 488-conjugated anti-mouse IgG were from Invitrogen. Vecta-shield with 4′,6-diamidino-2-phenylindole (DAPI) was from Vector Laboratories. Gel electrophoresis reagents were from Bio-Rad.