Journal: bioRxiv
Article Title: Progressive suppression of DNA repair genes with persistent p53 activation in Doxorubicin-treated cardiomyocytes
doi: 10.64898/2026.02.03.703628
Figure Lengend Snippet: (A) Experimental design of this study. iPSCs derived from three females and three males aged between 18-32 were differentiated into cardiomyocytes (iPSC-CMs). iPSC-CMs were treated for 24 hours with 0.5 μM Doxorubicin (DOX) and a Vehicle control (VEH) then assayed directly following treatment (tx+0), after 24 hours of recovery (tx+24), and after 144 hours of recovery (tx+144). (B) Purity of differentiated iPSC-CMs as measured by flow cytometry for cardiac troponin T (TNNT2) expression. (C) Percent viability of iPSC-CMs over increasing concentrations of DOX after 24 hours of treatment. Cell viability after DOX treatment was assessed in three individuals (Individuals 2,3,5) and normalized to matched VEH. Dose-response curves were generated using a four-point log-logistic regression with the upper asymptote set to 1. LD 50 values were calculated for each individual, with the median LD 50 shown in red. The selected sub-lethal concentration for further experimentation is represented by the grey line. (D) Quantification of DNA damage as measured by γH2AX expression intensity in DOX- and VEH-treated iPSC-CMs from three individuals (Individuals 1, 3, 4). Asterisks represent a statistically significant difference in γH2AX expression between DOX- and VEH-treated cells at each timepoint, as well as a statistically significant difference between DOX-treated cells across timepoints (* P < 0.05, ** P < 0.01, *** P < 0.001, t-test). (E) Immunofluorescence staining for the γH2AX marker (red) in iPSC-CM nuclei stained with Hoechst (blue) at tx+0, tx+24, and tx+144 in DOX- and VEH-treated cells in a representative individual (Individual 2). Scale bar of 100 μm.
Article Snippet: On Day 20 purified iPSC-CMs were dissociated from the culture plate with 0.05% trypsin/0.53 mM EDTA (MT25052CI, Corning) and counted using a Countess 2 machine. iPSC-CMs were plated in a galactose-containing, glucose-free Cardiomyocyte Maintenance Media (CMM) (500 mL DMEM without glucose (A14430-01, ThermoFisher Scientific), 50 mL FBS (MT35015CV, Corning), 990 mg Galactose (G5388, Sigma-Aldrich), 5 mL 100 mM sodium pyruvate (11360-070, ThermoFisher Scientific), 2.5 mL 1 M HEPES (H3375, Sigma Aldrich), 5 mL 100X GlutaMAX (35050-061, ThermoFisher Scientific), and 5 mL Penicillin/Streptomycin (100X)(30-002-CI, Corning) to metabolically mature iPSC-CMs. iPSC-CMs were maintained in culture for an additional 5-10 days.
Techniques: Derivative Assay, Control, Flow Cytometry, Expressing, Generated, Concentration Assay, Immunofluorescence, Staining, Marker
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