Fig. 5

U4-I05 enhances the sensitivity of tumor cells to oxaliplatin. (A) SW620 and LoVo cells were treated with oxaliplatin and/or U4-I05 (5 µM or 10 µM) for 72 h, and cell confluency was monitored using the Incucyte system. Inhibition curves were plotted, and statistical analysis was performed using a two-way ANOVA. U4-I05 enhanced the cytotoxic effect of oxaliplatin in both cell lines. Mean ± SD, n = 3. **p < 0.01, ***p < 0.001. (B) SW620 and LoVo cells were treated with U4-I05 (5 µM or 10 µM) in the presence or absence of oxaliplatin (50 µM) for 72 h, followed by crystal violet staining for colony counting. Mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. (C-D) SW620 and LoVo cells were treated with U4-I05 with or without oxaliplatin (50 µM) for 72 h. Apoptosis was evaluated using flow cytometry with PI staining (C) or Annexin V/7-AAD staining (D). U4-I05 treatment increased the proportion of PI + cells following oxaliplatin exposure. (E-H) HCT116 and HT-29 cells were treated with oxaliplatin and/or U4-I05 (5 µM or 10 µM) for 72 h. Cell confluency was monitored with the Incucyte system (E), and crystal violet staining was performed (F). Apoptosis was assessed using PI staining (G) or Annexin V/7-AAD staining (H). Inhibition curves were plotted, with statistical analysis conducted using a two-way ANOVA (E). U4-I05 did not affect oxaliplatin sensitivity in HCT116 and HT-29 cells. Mean ± SD, n = 3; ns, not significant. (I) CD44⁺ cells were sorted from SW620 and LoVo cells via flow cytometry and treated with U4-I05 (10 µM) and oxaliplatin (50 µM). Apoptosis was assessed using Annexin V/7-AAD staining, and the apoptosis rate was calculated. U4-I05 significantly increased the apoptosis rate in CD44⁺ cells exposed to oxaliplatin. Mean ± SD, n = 3. ***p < 0.001. (J-K) Oxaliplatin-resistant (OxaR) (J) and 5-Fu-resistant (5-FuR) (K) DLD-1 cells with USP4 knockdown were treated with or without U4-I05 (10 µM) and exposed to oxaliplatin (J) or 5-Fu (K) for 72 h in the Incucyte system. Cell confluency and IC50 values were assessed. U4-I05 treatment enhanced sensitivity to oxaliplatin and 5-Fu in resistant cells, but this effect was absent in USP4 knockdown cells. Mean ± SD, n = 3. ***p < 0.001. (L) The mRNA expression levels of PARP1, TP53, and ABCB1 were analyzed by RT-qPCR in 5-FuR DLD-1 cells and in 5-FuR DLD-1 cells with USP4 knockdown. Knockdown of USP4 reduced the mRNA levels of PARP1 and TP53 but did not affect ABCB1. Mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant