Fig. 8

Sec62 expression is upregulated by the METTL3-induced m⁶A modification. a Anti-m⁶A IP was performed with cell lysates of NCM460, DLD1 or SW480 cells using anti-m⁶A antibody. RT-qPCR was performed to detect Sec62 mRNA in the elutes. b DLD1 or SW480 cells were transfected with the indicated siRNAs. RT-qPCR was performed for evaluating the indicated genes. c DLD1 or SW480 cells were transfected with the indicated siRNAs. Western blot was performed using the indicated antibodies. d DLD1 cells were transfected with the indicated siRNAs. Me-RIP was performed using anti-m⁶A antibody in the cell lysates. RT-qPCR was performed to detect Sec62 mRNA in the elutes. e DLD1 or SW480 cells were transfected with the indicated plasmids. Western blot was performed using the indicated antibodies. f DLD1 cells were transfected with siRNAs or plasmids as indicated. Cells were treated with 2.5 μM actinomycin D and mRNA levels of Sec62 were analyzed by RT-qPCR (n = 3). g RIP experiments were performed with cell lysates from DLD1 or SW480 cells using anti-IGF2BP1 antibody. Coprecipitated RNA was purified and Sec62 mRNA was analyzed by RT-qPCR. h DLD1 cells were transfected with indicated siRNAs. Western blot was performed using the indicated antibodies. i DLD1 cells were transfected as indicated. After treatment with actinomycin D for indicated times, the decay of Sec62 mRNA was analyzed (n = 3). j DLD1 cells were transfected as indicated. RIP was performed and Sec62 mRNA level in the elutes was analyzed by RT-qPCR. k Total RNAs were extracted for CRC tissues and RT-qPCR was performed for the indicated genes. The correlations between Sec62 mRNA level and METTL3 mRNA level or IGF2BP1 mRNA level were plotted (n = 40). l A working model explaining how the m⁶A modification-mediated Sec62 induction promotes stemness of human colorectal cancer cell