Fig 2: Knockdown of METTL3 induces DNA damage accumulation and sensitizes colorectal cancer cells to 5-FU. (A) Cell survival analysis in METTL3-KD and control HCT-8 cells with 5-FU treatment. (B) Cell apoptosis was analyzed by flow cytometry. (C) Knockdown of METTL3 enhanced the expression of pro-apoptotic proteins caspase-3 and Bax in HCT-8 cells treated with 5-FU. (D) Morphological analysis of control and METTL3-KD HCT-8 cells with various doses (0, 2 and 4 µg/ml) of 5-FU treatment for 24 h. (E) Western blotting to determine ?-H2AX levels in control and METTL3-KD HCT-8 cells following various doses (0, 2 and 4 µg/ml) of 5-FU treatments for 4 h. (F) Immunofluorescence staining of ?-H2AX foci in different cells treated with 5-FU for 4 h. The quantification of the foci numbers per cells are presented on the right panel. Data are presented as the means ± SD. *P<0.05, **P<0.01 and ***P<0.001. METTL3, methyltransferase-like 3; 5-FU, 5-fluorouracil; KD, knockdown; sh-, small hairpin; CN or Con, control.

Fig 3: Inhibition of RAD51AP1 overcomes 5-FU-resistance in colorectal cancer cells. (A) Western blot analysis of RAD51AP1 in control and RAD51AP1-KD HCT-8R cells. (B) Cell survival assay in control and RAD51AP1-KD HCT-8R cells. (C) Morphological analysis of control and RAD51AP1-KD HCT-8R cells with 60 µg/ml 5-FU treatment for 48 h. (D) Knockdown of RAD51AP1 promotes apoptosis in HCT-8R cells with 5-FU treatment. (E) Silencing of RAD51AP1 induces caspase-3 and Bax expression in HCT-8R cells following 5-FU treatment. (F) Western blotting to determine ?-H2AX levels in control and RAD51AP1-KD HCT-8R cells following 5-FU treatments for 4 h. (G and H) Immunofluorescence staining of ?-H2AX foci and 53BP1 foci in different cells treated 5-FU for 4 h. (I) Comet assay of RAD51AP1-KD and control HCT-8R cells treated with or without 5-FU (60 µg/ml) for 12 h. *P<0.05, **P<0.01 and ***P<0.001. RAD51AP1, RAD51 associated Protein 1; 5-FU, 5-fluorouracil; KD, knockdown; HCT-8R, 5-FU-resistant HCT-8 cells; sh-, small hairpin; CN, control.

Fig 4: Knockdown of RAD51AP1 promotes DNA damage and sensitizes HCT-8 cells to 5-FU. (A) Western blotting identified the knockdown of RAD51AP1 in HCT-8 cells. (B) A cell survival assay demonstrated that downregulation of RAD51AP1 sensitizes HCT-8 cells to 5-FU. (C) Cell apoptosis analyzed by flow cytometry in control and RAD51AP1-KD HCT-8 cells with 4 µg/ml 5-FU treatment for 24 h. (D) Knockdown of RAD51AP1 promoted the expression of caspase-3 and Bax in HCT-8 cells treated with 4 µg/ml 5-FU for 12 h. (E) Western blotting to determine ?-H2AX levels in control and RAD51AP1-KD HCT-8 cells following 4 µg/ml 5-FU treatments for 4 h. (F and G) Immunofluorescence staining of ?-H2AX foci and 53BP1 foci in different cells treated 4 µg/ml 5-FU for 4 h. (H) Comet assay of RAD51AP1-KD and control HCT-8 cells treated with or without 5-FU. *P<0.05, **P<0.01 and ***P<0.001. RAD51AP1, RAD51 associated Protein 1; 5-FU, 5-fluorouracil; KD, knockdown; sh-, small hairpin; CN, control.