Harmony 3.5.1 high content imaging and analysis software

The pCDH-GFP-NONO-viral vector was used to infect MDA-MB-231 cells, which were seeded at a density of 1×10³ cells/well in a 96-well plate. After 24 h of incubation, the cells were exposed to the drug library compounds (BML-2843-0100; Enzo) at a final concentration of 5 µM in 0.5% dimethyl sulfoxide (DMSO) (v/v) (Sigma-Aldrich, St Louis, MO). After a 24 h incubation, the cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) and washed with Dulbecco's phosphate-buffered saline (DPBS; Welgene; Korea) for 10 min. GFP intensities were determined with the Operetta High Content Screening System (Perkin Elmer, Waltham, MA) and analyzed by Harmony 3.5.1 high content imaging and analysis software (Perkin Elmer).

1.5×10³ cells/well of Fa2N-4 and 0.8×10³ cells/well of Huh7 were mixed and plated in 384 well plate. After 16 h, 10 μM of 43 compounds (see Additional file 1: Figure S5) were treated to each well in duplicate. After 3 days incubation, cells were fixed with 4 % PFA and permeabilized with 0.1 % Triton X-100. The cells were reacted with primary anti-CHALV1 and AFP for 16 h at 4 °C and washed with PBS for 10 min, and were incubated for 2 h at room temperature with goat anti-rabbit Alexa Fluor® 633 and goat anti-mouse Alexa Fluor® 488 secondary antibodies. After three times washing with PBS, cells were stained with Hoechst 33342 for nucleus. Cell images were obtained by Operetta® High Content Screening System and analyzed by Harmony 3.5.1® high content imaging and analysis software (Perkin Elmer).
Sorafenib was treated as positive control and Z’ score was calculated by Harmony High-Content Imaging and Analysis Software using positive and negative (DMSO treatment) control. The compounds that > 50 % Huh7 and < 20 % Fa2N-4 inhibition were selected.