Fig 2: Substance testing of Penicillin G (PenG) and 5-Fluorouracil (5-FU) in the human induced pluripotent stem cell Test (hiPS Test). Human iPSCs were differentiated into cardiomyocytes over a time course of 10 days (Figure 3A). On day 10, endpoints were analyzed. (A): Pipetting scheme for compound testing in the hiPS Test. C6-C1: Increasing concentrations of a tested substance in quadruplicates, SC: solvent control, LC: lysis control, BC: background control, white circles: border wells filled with 800 µL sterile water. (B–E): Cells were treated with 1.2–300 µM PenG or 0.012–3 µM 5-FU for 10 days. Substances were freshly applied on every feeding day (days 2, 3, 4, 6, and 8). (B,C): Concentration response curves normalized to the control of cell viability, beating frequency, and beating area for PenG and 5-FU, respectively, including the benchmark response (BMR)20 and BMR30. On day 10 videos of every well were recorded using a Binocular (Leica, Wetzlar, Germany, DS100B) with an integrated heating plate set to 37 °C. Video analyses were performed with the in-house developed software CardioVision to evaluate the beating frequency and beating area. A cell viability assay was performed and analyzed using a multimode-microplate reader (TECAN, Männedorf, Switzerland, Infinite® 200 PRO). (D,E): Samples were collected (quadruplicates) and pooled for RT-qPCR analysis of TNNT2, an early cardiomyocyte marker, and ACTN2, a later cardiomyocyte marker. Mean values of copy numbers of the target genes TNNT2 and ACTN2 were normalized to the reference gene CANX which is stably expressed in hiPSCs and cells of the mesodermal lineage [59,60] including early cardiomyocytes in the hiPS Test (Figure S2). For all experiments, N = 3, ±SEM, p < 0.05 was considered significant, * = significant compared to the solvent control. N/A = not applicable.