(A) Schematic diagram showing the timing of the FUCCI2a fluorescence reporters. Red indicates mCherry; green indicates mVenus. Dot plots show the cell-cycle profile and proportion of FUCCI RPTECs in G₁, early S, and S/G₂/M phases, as analyzed by flow cytometry. Primary RPTECs from FUCCI2a-gGT-Cre mice were synchronized in 1% FBS PTC media and then treated with cisplatin, with or without VE-821, or were left untreated for 48 hours. Cells were then fixed and analyzed by flow cytometry. Red and green colors in the graph indicate G₁ and S/G₂/M phases, respectively, and double-positive cells were considered to be in the early S phase. RFP, red fluorescent protein; GFP, green fluorescent protein. (B and C) Cell-cycle assessment by live microscopy of HK2 cells expressing the FUCCI system in control conditions (Ctrl, n = 4), or incubated with 50 nM pifithrin-α (PF50, n = 2) or 10 nM rigosertib (RG10, n = 2). (B) Representative images of the cells at 1 hour and 24 hours after incubation are shown. Images on the left show overlays of bright-field, monomeric Kusabira-Orange (mKO), and monomeric Azami-Green (mAG) channels. Images on the right show mKO and mAG channels (original magnification, ×10). (C) Graph shows the quantification over time of the cells in G₁ (mKO-Cdt1, red), G₂ (mAG-geminin, green), and S (mKO-Cdt1⁺ mAG-geminin, orange) phases of the cell cycle. (D) Cell-cycle analysis by propidium iodide staining and flow cytometry of mouse primary cells at baseline (top 2 graphs) and after treatment with cisplatin at 0.2 μg/mL for 48 hours (bottom 2 graphs). (E and F) Results of the cell-cycle analysis for 3 independent experiments. Data are presented as the mean ± SEM. Statistical significance was determined by 1-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 and **P < 0.01.