Additional file 4: of A kinase inhibitor screen identifies a dual cdc7/CDK9 inhibitor to sensitise triple-negative breast cancer to EGFR-targeted therapy

Figure S2. EGFR-negative TNBC cell line SUM185PE is insensitive to co-treatment with lapatinib and PHA-767491. a. Immunofluorescence imaging of EGFR-positive (SKBR7) and EGFR-negative (SUM185PE) TNBC cell lines. Cells were fixed using 1% paraformaldehyde and 0.1% Triton-X for 15 min before being washed thrice with 1x PBS and blocked with 0.5% bovine serum albumin in PBS for 30 min. EGFR antibody (sc-03; Santa Cruz Biotechnology®) was used to stain EGFR overnight at 4 °C. Fixed cells were then incubated with anti-rabbit Alexa 488-conjugated secondary antibody (A11008; Molecular Probes®) or Hoechst (nuclear stain; 1:10,000) for 1 h at room temperature in the dark before being imaged at 20x magnification. b. Impact of EGF stimulation on EGFR-mediated signal transduction in EGFR-negative cell line SUM185PE. Cells were starved overnight in serum-free medium before being treated with lapatinib (3.16 μM) for 4 h and subsequently stimulated with EGF (100 ng/ml) for 5 min. Cells were then lysed and protein samples subjected to SDS-PAGE and immunoblotting with indicated antibodies. c. Dose-response experiment combining lapatinib and PHA-767491 in SUM185PE cells. The upper graph shows the response of SUM185PE cells to lapatinib and PHA-767491 monotherapies. The lower graph displays the proliferation of SUM185PE cells after combining lapatinib (3.16 μM) with dose range (0.01–10 μM) of PHA-767491. Proliferation was assessed using sulphorhodamine B assay and results were normalised to DMSO using the % control method as outlined in materials and methods. (PDF 525 kb)