Columbus image data storage and analysis system

The assay is described in detail elsewhere [30 (link)]; briefly, Huh 7-hACE2 cells were seeded overnight to adhere to a 96-well plate, treated with serial dilution of the compounds, and then infected with SARS-CoV-2 with appropriate controls (vehicle, not infected, infected and not treated, and infected and treated with the control inhibitor hydroxychloroquine). The plates were incubated for 20 h at 37 °C, and then fixed with 4% PFA, permeabilized with 0.1% of Triton-X for 15 min and incubated in blocking buffer (PBS containing 1% of BSA).
The antibody mSIP-3022 was diluted in blocking buffer and incubated for 2 h at 37 °C. The cells were washed twice in PBS and incubated with the secondary antibody AlexaFluor488-conjugated goat anti-mouse IgG (Cat No. A-11001, ThermoFisher, Rockford, IL, USA) plus DAPI for 1 h at 37 °C. Each plate was kept in PBS after washing. Digital images were acquired using the Operetta high content imaging system (Perkin Elmer, Walthem, MA, USA). The digital images were taken from nine different fields of each well. The total number of cells (nuclei) and the number of infected cells were analysed using the Columbus Image Data Storage and Analysis System (Perkin Elmer, Waltham, MA, USA).

A-673 and SJCRH30 cells were plated into black 96-well cell carrier plates (PerkinElmer) at a density of 3500 cells/well and allowed to adhere overnight. The cells were treated in triplicate with DMSO or the indicated concentrations of ATXII for 6 or 24 h, then fixed with paraformaldehyde. After fixation, the cells were incubated in a blocking solution of 10% bovine calf serum in DPBS for 20 min at room temperature. The cells were then incubated in a primary antibody against γ-H2A.X (1:400; Cell Signaling Technology) diluted in 1% bovine serum albumin/0.3% Triton X-100 in DPBS, overnight, at 4 °C. The cells were subsequently washed with DPBS and incubated with an Alexafluor-594-conjugated secondary antibody (1:1000; Life Technologies) for 1 h at room temperature. The plates were washed with DPBS, and the nuclei stained with NucBlue live cell stain (Life Technologies) diluted in DPBS. Images were collected using an Operetta high-content imaging system (PerkinElmer) using a 20× long working distance objective and analyzed with the Columbus Image Data Storage and Analysis System (PerkinElmer). A minimum of three fields were collected per well, with all concentrations tested in triplicate.

Cells were seeded in 384-well plates at 1,000 cells/well, treated with LCL161 + GEM/CIS for 5 days, stained with DAPI and imaged using an Operetta CLS™ high-content system. The nuclei number was analyzed using a Columbus Image Data Storage and Analysis System (PerkinElmer). The combination index (CI) was calculated using CompuSyn software version 1.0 (ComboSyn, Inc., Paramus, NJ, USA) and Chou-Talalay’s equation (19 (link)). The CI < 1, CI =1, and CI > 1 indicate synergism, additive effect, and antagonism, respectively.