Cellinsight high content microscope

MFC10A parental cell lines were grown according to published protocols (38 (link)). Derivative isogenic cell lines were generated though stable infection using viral infection of cell pools using the indicated vectors (Supplementary Table S1). Control MCF10A cell lines were generated by expressing empty vectors conferring puromycin, or blasticidin gene resistance as appropriate. Proliferation was measured by staining with Hoescht nuclear dye and cell (nuclear) number counted using a Thermo CellInsight high content microscope. The parental cell line was first screened against all 90 compounds (Selleckchem, Houston, TX) to determine concentration-response curves and approximate IC₅₀ concentrations (Supplementary Table S2). The maximum concentration assayed for any drug was approximately 20μM. Each line was independently screened by plating 1,000 cells/well in 384-well plates for 24 hours then exposed to each drug at IC₅₀ concentration for 72 hours using a minimum of 8 replicates. Statistical scoring is described in detail in the supplemental methods.

H1975-RR cells were seeded in 384-well plates at a density of 1,000 cells/well in the presence of 2uM rociletinib or vehicle and after 24 hours exposed to three different doses of compounds from a 90-drug library for 72 hours. At the end of this period nuclei were stained with Hoechst 33422 (Life Technologies) and counted using a Thermo CellInsight high content microscope. The screen was repeated three times using varying library concentrations of 5 μg/mL, 500 ng/mL, and 50 ng/mL and each combination measured in quadruplicate. Raw cell numbers were median normalized on a per-plate basis. For each compound in the library, the relative cell number in the DMSO plate was compared with the number in the rociletinib plate using a Student’s t-test. A synergy score was developed based on the −log10 of the P value of this t-test and was signed to indicate synergistic inhibition of growth (positive score) or antagonism (negative score). The reported synergy score is based on the average of scores over three different library concentrations.

Small molecule inhibitors were all purchased commercially from Selleck Chemicals, and included Osimertinib (S7297), Alectinib (S2762), XAV-939 (S1180), and PRI-724 (S8262). Dimethyl sulfoxide (DMSO) (Fisher Scientific) was used to dissolve small molecule inhibitors according to manufacturer’s recommendations for use in in vitro experiments. PC9 and H3122 cells (5 × 10³) were seeded in 96-well plate format (jclear CellStar, Greiner) and rested for 24 hours before treatment. Treatment included: i) DMSO, ii) tyrosine kinase inhibitors (TKI) Osimertinib (PC9 cells) or Alectinib (H3122 cells), iii) Wnt/β-catenin inhibitors PRI-724 or XAV-939, and v) indicated combination therapies of TKI and Wnt/ β-catenin inhibitors. All conditions were plated in technical quadruplicate and cells were retreated every 3 days. At each imaging interval, cellular nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) and scanned using a CellInsight High-Content Microscope (Version 6.4.3 Build 7204, Thermo Fisher Scientific) with a 4X objective.