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. 2026 Jan 14;34(1):201129. doi: 10.1016/j.omton.2026.201129

Figure 2.

Figure 2

Treatment with pyrimidine-based compounds induces phenotypic hallmarks of senescence in cancer cells

(A) Cell cycle was performed after treatment with 5 μM of P12 and P14. Cells were cultured until they reached approximately 80% confluency. At that point, 10 μM of 5-ethynyl-2′-deoxyuridine (EdU) was added to the medium to label cells actively synthesizing DNA during the S phase. Following a 2-h incubation at 37°C to enable EdU incorporation, cells were collected and fixed. This was done by suspending the cells in PBS, centrifuging at 500 × g for 5 min, and then resuspending the pellet in 500 μL of 70% ethanol for fixation. MCF7 UT (G0/G1: 11.65%, S: 61.1%, G2/M: 26.25%); MCF7 P12 (G0/G1: 20.35%, S: 63.8%, G2/M: 15.65%); MCF7 P14 (G0/G1: 15.9%, S: 68.15%, G2/M: 13.1%); A375 UT (G0/G1: 34.45%, S: 57.45%, G2/M: 5.44%); A375 P12 (G0/G1: 25.4%, S: 72.9%, G2/M: 1.94%); A375 P14 (G0/G1: 33.2%, S: 67.3%, G2/M: 0.52%). (B) Heatmap showing the mRNA levels of different cell-cycle regulators in MCF7 and A375 cells after treatment with 5 μM of P12 and P14 for 7 days. (C) qPCR analysis showing the expression levels of the senescent markers p21, p53, and p53-related genes IGFBP3 and GDF15 in MCF7 cells (left) and A375 (right) after treatment with 5 μM of P12 and P14 for 7 days. The mean of three independent experiments is shown. (D) Representative images of the SA-β-galactosidase activity in cancer cells after treatment with P12 and P14. Quantifications are shown in the right. (E) Heatmap showing the mRNA levels of SASP factors (IL-6, IL-8, MMP3, and MMP9) in MCF7 and A375 cells after treatment with 5 μM of P12 and P14 for 7 days. Data represent the mean ± SEM of three independent experiments. (F) Flow cytometry in MCF7 and A375 after treatment with 5 μM of P12 and P14 for 7 days. Cells were stained using a dual labeling approach with PI at 2 μg/mL and YO-PRO-1 at 150 nM. Cells negative for both PI and YO-PRO-1 were classified as viable, while apoptotic stages were determined according to the levels of YO-PRO-1 or PI incorporation. Viable cells were identified using forward scatter (FSC) and side scatter (SSC) parameters to exclude cellular debris. Additionally, single cells were separated from aggregates based on FSC-H and FSC-A gating (n = 3). (G) Graphical abstract illustrating the effects of pyrimidine compounds in the different cellular models. Data are presented as mean ± SEM. Two-tailed Student's t test and one-way ANOVA were used to calculate the significance represented as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001.