Fig 1: Overexpression of KCNQ1OT1 alleviated the inhibitory effect of RBM15 knockdown on DDP resistance in LC cells by inhibiting iron death. oe-KCNQ1OT1 was transfected into drug-resistant cells, with transfection of oe-NC as a negative control. (A): The cell inhibition rate of LC cancer cell lines treated with DDP was measured by CCK-8 assay. The cells were treated with 10 µg/mL DDP, and then (B): The number of cell clones was determined by colony formation assay; (C): The intracellular ROS levels were measured using the DCFH-DA probe; (D): The MDA levels in cells were evaluated using a lipid oxidation assay kit; (E): The GSH content in cells was assessed using a GSH assay kit; (F): The iron content in cells was measured by colorimetric assay; (G): The expression of ACSL4 in cells was detected by Western blot. The experiments were independently repeated three times, and the data are expressed as mean ± standard deviation. Data comparisons among multiple groups were performed using two-way ANOVA, followed by Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01
Fig 2: RBM15 promoted DDP resistance in LC cells. (A-B): The expression of RBM15 in 16HBE cells and LC cancer cells was detected by RT-qPCR and Western blot assay; (C): Cell inhibition rate of LC cancer cell lines treated with DDP was measured by CCK-8 assay; (D): The number of cell clones after treatment with 10 µg/mL DDP was determined by colony formation assay. si-RBM15 was transfected into drug-resistant cells, with transfection of si-NC as a negative control, and then: (E-F): The expression of RBM15 in drug-resistant cells was detected by RT-qPCR and Western blot; (G): Cell inhibition rate of LC cancer cell lines treated with DDP was measured by CCK-8 assay; (H): The number of cell clones after treatment with 10 µg/mL DDP was determined by colony formation assay. The experiments were independently repeated three times, and the data are expressed as mean ± standard deviation. Data in panels A, B, and D: comparisons among multiple groups were analyzed using one-way ANOVA; data in panels C and E-H: comparisons among multiple groups were analyzed using two-way ANOVA. Tukey’s multiple comparisons test was used for post hoc analysis. In panels A-B, * p < 0.05 compared to 16HBE cells, ** p < 0.01 compared to 16HBE cells, ## p < 0.01 compared to the parental cells; in other panels, ** p < 0.01
Fig 3: RBM15 is highly expressed in NSCLC. A-B: The expression of RBM15 in lung squamous cell carcinoma and lung adenocarcinoma was predicted using the Starbase and UALCAN databases. C-D: The expression of RBM15 in tumor tissues and adjacent non-tumor tissues (control) in NSCLC patients was detected by RT-qPCR and Western blot assay (representative bands), N = 50; the comparison between the two groups in panels C-D was performed using paired t-test. E–F: The expression of RBM15 in different cells was detected by RT-qPCR and Western blot assay, with 16HBE as control, N = 3; the comparisons among multiple groups in panels E–F were conducted using one-way ANOVA, followed by Tukey's multiple comparisons test. The data were presented as mean ± standard deviation. ** p < 0.01
Fig 4: KLF1 promotes the transcription level of ANXA8. A-B: The expression of ANXA8 in tumor tissues and adjacent non-tumor tissues (control) in NSCLC patients was detected by RT-qPCR and Western blot assay (representative bands), N = 50; the comparison between two groups in panels A and B was analyzed using paired t-test. C-D: The expression of ANXA8 in different cells was detected by RT-qPCR and Western blot assay, with 16HBE used as control, N = 3; the comparison among multiple groups in panels C and D was analyzed using one-way ANOVA, followed by Tukey's multiple comparisons test. E: The binding sequence of KLF1 to the ANXA8 promoter was predicted by the JASPAR database. F: The enrichment of KLF1 on the ANXA8 promoter in cells was analyzed by ChIP, with si-NC or si-RBM15-3 + oe-NC used as control, N = 3; the comparison among multiple groups in panel F was analyzed using two-way ANOVA, followed by Tukey's multiple comparisons test. G: The binding of KLF1 to the ANXA8 promoter was analyzed by the dual-luciferase reporter assay, with oe-NC used as control, N = 3; the comparison among multiple groups in panel G was analyzed using two-way ANOVA, followed by Tukey's multiple comparisons test. H: The transcription levels of ANXA8 in different cells were detected by RT-qPCR, with si-NC or si-RBM15-3 + oe-NC used as control, N = 3; the comparison among multiple groups in panel H was analyzed using one-way ANOVA, followed by Tukey's multiple comparisons test. I: The correlation between ANXA8 and KLF1 and RBM15 in NSCLC tumor tissues was analyzed by Pearson correlation analysis, N = 50. The data were presented as mean ± standard deviation. ** p < 0.01
Fig 5: FER1L4 silencing alleviated the inhibitory effect of RBM15 knockdown on DDP resistance in LC cells by inhibiting ferroptosis. si-FER1L4 was tranfected into drug-resistant cells, with transfection of si-NC as a negative control. (A): The expression of FER1L4 in cells was detected by RT-qPCR; (B): The cell inhibition rate of LC cancer cell lines treated with DDP was measured by CCK-8 assay; The cells were treated with 10 µg/mL DDP, and then (C): The number of cell clones was determined by colony formation assay; (D): The intracellular ROS levels were measured using the DCFH-DA probe; (E): The MDA levels in cells were evaluated using a lipid oxidation assay kit; (F): The GSH content in cells was assessed using a GSH assay kit; (G): The iron content in cells was measured by colorimetric assay; (H): The expression of GPX4 in cells was detected by Western blot. The experiments were independently repeated three times, and the data are expressed as mean ± standard deviation. Data comparisons among multiple groups were performed using two-way ANOVA, followed by Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01
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