Fig 1: circCIMT interacts with APEX1 to suppress DNA damage. A) Mass spectrometry analysis of APEX1 on the circCIMT RNA pull-down assay samples. B) The interaction between circCIMT and APEX1 was confirmed by western blot analysis of the circCIMT pull-down samples. C) The RIP assay using an anti-APEX1 antibody was used to confirm that circCIMT and APEX1 interacted with each other. D) APEX1 expression in the lung tissue of untreated and Cd-treated mice was assessed by western blot analysis. n (Control) = 10, n (Cd) = 10. E) Correlation between APEX1 (APEX1/Tubulin) and circCIMT (2-?CT) in mouse lung tissue. F) ?-H2AX levels in si-APEX1-treated BEAS-2B and 16HBE cells were assessed by western blot analysis. G) ?-H2AX levels in APEX1-overexpressing BEAS-2B and 16HBE cells were examined by western blot analysis. H) Western blot analysis of APEX1 protein expression levels in 16HBE cells after simultaneous knockdown of circCIMT and APEX1. I) The number of ?-H2AX foci was detected by immunofluorescence staining after circCIMT and APEX1 co-interference. Scale bar = 5 µM. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: Simultaneous knockdown of circCIMT and APEX1 promotes expression of tumor‐associated genes and malignant transformation of cells. A) KEGG scatter plot of CSC‐like pathway gene sets in si‐circCIMT‐ versus si‐NC‐treated cells. B) Pik3cb, Myc, Cdkn1a, and Smad3 mRNA expression levels in human lung tissue. n (normal) = 19, n (cancer) = 14. C–F) Pik3cb, Myc, Cdkn1a, and Smad3 mRNA expression levels were detected in chronic Cd‐treated cells simultaneously treated with si‐circCIMT and si‐APEX1. G) The EdU assay was used to assess cell proliferation levels in chronic Cd‐exposed cells simultaneously treated with si‐circCIMT and si‐APEX1. H) Anchorage‐independent cell growth ability was measured in 16HBE cells continuously exposed to Cd for 25 weeks. I–K) Tumor volumes and weights in nude mice injected with knockdown of both circCIMT and APEX1 cells and exposed to Cd. n (NC) = 6, n (circCIMT sh) = 6, n (circCIMT sh+APEX1 sh) = 6. *p < 0.05, **p < 0.01, ***p < 0.001
Fig 3: circCIMT-interference inhibits the expression of nuclear base excision repair (BER) complex proteins after Cd exposure. A–C) Western blot analysis of ?-H2AX, APEX1, XRCC1, PARP1, and LIG3 protein expression levels in Cd-exposed cells following treatment with circCIMT#2- and circCIMT-overexpression vectors. D–F) Western blot analysis of APEX1, XRCC1, PARP1, and LIG3 protein expression levels in Cd-treated cells after circCIMT knockdown. G) Co-localization and expression of APEX1, XRCC1, PARP1, and LIG3 were detected in circCIMT-silenced cells exposed to Cd by immunofluorescence staining. Scale bar = 5 µM. H) Nuclear/cytoplasmic circCIMT levels were analyzed in control and Cd-treated cells. I) Western blot analysis of the nuclear/cytoplasmic protein levels of APEX1, XRCC1, PARP1, and LIG3 in Cd-exposed cells following circCIMT knockdown. *p< 0.05, **p < 0.01, ***p < 0.001.
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