2.2. Detect Gene Expression

For mRNA level examination, total RNA of BCa cells was extracted using Trizol reagent (Invitrogen). Complementary DNA (cDNA) synthesis was performed with the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, RR047A) using 1 μg RNA per sample. qPCR reactions were performed using TB Green® Premix Ex Taq™ (Takara RR820A) to determine mRNA transcript levels. Primers for qRT-PCR are listed in Supplementary Table S1, siRNAs are used to knockdown METTL3, and AFF4 expression is listed in Supplementary Table S2.

For Western blotting, BCa cells were lysed with RIPA buffer as a standard protocol. The cell lysate was then mixed with loading buffer and incubated at 100°C for 5 min and subjected to conventional Western analysis. Antibodies are listed in Supplementary Table S3. The relative levels of proteins were quantified using densitometry with the Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA). The target bands were densitometrically quantified and indicated under each band.