Spectramax i3 multi mode platform

The CellROX^® Green Reagent (Invitrogen, Carlsbad, California, USA) was used for oxidative stress detection. CellROX is a fluorogenic probe which is weakly fluorescent while in a reduced state. It exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA, with the absorption/emission maxima of 485/520 nm.
The cells were inoculated into a six‐well plate at a concentration of 2.5 × 10⁵ cells·mL⁻¹. The tested compounds were added when 70–80% confluence was achieved. After a 48‐h incubation, the cells were stained with the CellROX Orange Reagent and Hoechst 33342 in complete medium for 30 min at 37 °C. Next, the cells were washed with PBS and the fluorescence signal was measured by means of the SpectraMax i3 Multi‐Mode Platform (Molecular Devices, San Jose, California, USA). Each experiment was conducted three times with measurement in triplicate.

The determination of DSB was evaluated with a HCS DNA Damage Kit (Invitrogen, Carlsbad, USA) according to the manufacturer’s instruction. It is based on phosphorylated H2AX level measurement. The cells were seeded into 96-well plates in a concentration of 2 × 10⁴ cells/well. The tested compounds were added when 70–80% of confluence was achieved. After 48-h incubation, phosphorylated H2AX (Ser139), induced in response to double-strand breaks (DSBs) formation, was measured using specific primary antibody and Alexa Fluor™ 555 conjugated secondary antibody. The fluorescence signal was measured using the SpectraMax i3 Multi-Mode Platform (Molecular Devices, San Jose, USA).

The cells were seeded into 96-well plates in a concentration of 1.5 × 10⁵ cells/mL. The tested compounds (in a final concentration of 10, 125, and 250 µM) were added when 70%–80% of confluence was achieved. After 24-h incubation, the DNA damage (DSB) was determined using the HCS DNA Damage Kit (Thermofisher, Waltham, Massachusetts, USA) according to the manufacturer’s instruction. Phosphorylation of H2A histones is a known response to DSBs formation specifically. The DSBs level was measured by specific antibody-based detection of phosphorylated H2AX (Ser139) in the nucleus. The fluorescence of secondary antibody was measured using the SpectraMax i3 Multi-Mode Platform (Molecular Devices, San Jose, California, USA). Each experiment was conducted three times with measurement in triplicate.

Caspase 3/7 activity assay was performed using Caspase-Glo^® 3/7 Assay System (Promega Corporation, Madison, WI) according to manufacturer instructions. Cells were grown in Corning 96-well, clear bottom, white polystyrene plates (Corning, NY) at an approximate density of 15,000 cells/well. At the conclusion of the experiment, luminescence was measured on a SpectraMax i3 Multi-Mode Platform (Molecular Devices, Sunnyvale, CA).

Annexin V assay was performed using the RealTime-Glo^TM Annexin V Apoptosis Assay (Promega Corporation, Madison, WI) according to manufacturer instructions. Cells were grown in Corning 96-well, clear bottom, white polystyrene plates (Corning, NY) at an approximate density of 15,000 cells/well. Hourly luminescence measurements were measured on a SpectraMax i3 Multi-Mode Platform (Molecular Devices, Sunnyvale, CA).

Human iPSCs were washed with cold PBS, incubated for 10 min on ice in RIPA Buffer (Sigma-Aldrich), and supernatant collected. Three replicates were used for each condition. The supernatant protein content was determined using a Pierce BCA Protein Assay kit (Thermofisher Scientific) colorimetric reaction and quantified on a SpectraMax i3 Multi-Mode Platform (Molecular Devices). Subsequently, 20 μg of protein from each sample was resolved by SDS-PAGE, and transferred to a nitrocellulose membrane (Invitrogen). The membranes were incubated overnight at 4°C with primary antibodies: anti-ROCK1 (AbCAM 1:200), anti-CDH1 (AbCAM 1:200), anti-GAPDH, (Invitrogen 1:10,000), followed by incubation (30 min at room temperature) with infrared secondary antibodies: IRDye 800CW and IRDye 680CW (LI-COR 1:13,000), and imaged on the Odyssey Fc Imaging System (LI-COR Biosciences). Protein levels were quantified using Image Studio Lite (LI-COR Biosciences).