Incucyte

For siRNA experiments, cells were plated in 96-well plates at 6000 cells per well and treated with compounds 24 h later. The cells were transfected with 15 nM siRNA against KLF5 (ONTARGETplus SMART-POOL®, Dharmacon) or with a control siRNA at the respective concentration (ONTARGETplus SMART-POOL®, Dharmacon) using Lipofectamine® RNAiMAX (Invitrogen) following the manufacturer’s protocol. Alternatively, for siRNA experiments against NEK3 and TTC7A, and for additional KLF5 experiments, cells were plated in 6-well plates at 400,000 cells per well and treated with compounds 24 h later. The cells were transfected with 30 nM against NEK3 and TTC7A or with 5 nM and 10 nM against KLF5; 48 h post seeding, the cells were split and plated in 96-wells at 10,000 cells per well. The effect of the knockdown against KLF5, NEK3, or TTC7A on cell viability/proliferation was measured by live cell imaging using Incucyte® (Satorius) or by the MTT assay (Sigma M2128) at 72 h (or at the indicated time point) post-transfection; 500 µg/mL MTT salt was diluted in complete medium and incubated at 37°C for 2 h. Formazan crystals were extracted using 10% Triton X-100 and 0.1 N HCl in isopropanol, and color absorption was quantified at 570 nm and 630 nm (Alliance Q9, Uvitec).

Cellular proliferation was evaluated using Incucyte^®, a live-cell analysis and imaging system (Satorius, Ann Arbor, MI, United States). Approximately 2.5 × 10³ MSC/well were seeded in a 96-well plate and allowed to propagate in culture for 72 h. MSC migratory function was tested using a QCM^TM Colorimetric Cell Assay (EMD Millipore, Burlington, MA, United States; Cat.# ECM508), performed according to the company’s standard protocol.