Fig 2: NAC attenuates BPAF-induced apoptosis and cell cycle arrest in ST cells. (A) Apoptotic rates in ST cells were analyzed via Annexin V-FITC/PI staining and flow cytometry post-treatment in the CON, BG, and NBG groups. (B) Quantitative assessment of apoptosis following the treatments. (C,D) The expression levels of BAX, BAD, and BCL-2 were determined using RT-qPCR (C) and western blot (D) across the treatment groups. (E) Quantitative representation of the western blot results in bar graph format. (F) Cell cycle phase distribution post-treatment, determined through flow cytometry, indicating shifts in cell cycle dynamics. (G) Histogram and statistical analysis illustrating the cell cycle alterations. (H,I) The expression levels of PCNA, CDK2, and CDK4 were determined using RT-qPCR (H) and western blot (I) across the treatment groups. (J) Quantitative representation of the western blot results in a bar graph format. 0 μM BPAF + 0 mM NAC group, CON; 50 μM BPAF + 0 mM NAC group, BG; 50 μM BPAF + 5 mM NAC group, NBG. The data presented here represent the mean ± SD of at least three separate experiments. * p < 0.05 and ** p < 0.01 versus the CON. # p < 0.05 and ## p < 0.01 versus the BG. ns, not significant (p > 0.05).

Fig 3: The association of LTB4R with BAX, BCL2, CASP3, CCND1, CDK2, and CDK4. (A–F) The difference of BAX, BCL2, CASP3, CCND1, CDK2, and CDK4 scores defined by using ssGSEA between LTB4R high expressed group and LTB4R low expressed group. In addition, the association of LTB4R expression with BAX, BCL2, CASP3, CCND1, CDK2, and CDK4 scores. Wilcoxon rank-sum test, * p < 0.05.

Fig 4: Effects of BPAF on apoptosis and cell cycle in ST cells. (A) Flow cytometry analysis of ST cell apoptosis using Annexin V-FITC/PI staining following 50 μM BPAF exposure (B) Quantification of apoptosis rates in ST cells after 50 μM BPAF treatment. (C,D) The expression levels of BAX, BAD, and BCL-2 were determined using RT-qPCR (C) and western blot (D) after 50 μM BPAF treatment. (E) The western blot quantification is shown as a bar graph. (F) Flow cytometry was utilized to determine the relative proportion of cells with each cell cycle phase after 50 μM BPAF treatment. (G) Histogram and statistical analysis for cell cycle distribution in ST cells. (H,I) The expression levels of PCNA, CDK2, and CDK4 were determined through RT-qPCR (H) and western blot (I) after 50 μM BPAF treatment. (J) The western blot quantification is shown as a bar graph. The data presented here represent the mean ± SD of at least three separate experiments. * p < 0.05 and ** p < 0.01 versus 0 μM. ns, not significant (p > 0.05).

Fig 5: Interfering with LTB4R inhibited ccRCC cell cycle and promoted ccRCC cell apoptosis. (A,B) Knockdown of LTB4R promoted apoptosis in Caki1 and ACHN cells. (C–F) After knockdown of LTB4R, the proportion of the G1/G0 phase in Caki1 and ACHN cells increased, while the proportion of the G2/M phase decreased, and the proportion of the S phase did not change significantly. (G) Western Blot Analysis showed that after knockdown of LTB4R, the apoptosis-related proteins, BAX and c-CASP3 (c-caspase-3) were increased and the anti-apoptotic protein BCL2 was decreased in Caki1 and ACHN cells. (H) Western Blot Analysis showed that the period-related proteins CCND1 (CyclinD1), CDK2, and CDK4 were all reduced after LTB4R knockdown. A 2-tailed t test was used (* p < 0.05; ** p < 0.01, *** p < 0.001, ns, not significant).