Figure 6.

hnRNPA2/B1 controls the dynamic sorting of m⁶A-modified Circ-CDYL into exosomes

(A) RNA antisense purification (RAP) assays using a biotinylated antisense probe of Circ-CDYL or negative control probe were performed using Circ-CDYL-overexpressing HCC cells (SMMC-7721 Ov-Circ-CDYL) (left), and m⁶A sites-mutated Circ-CDYL-overexpressing HCC cells (SMMC-7721 Ov-Circ-CDYL^Mut) (right), followed by Western blot assays using an anti-hnRNPA2/B1 antibody.

(B) RIP assays using an anti-hnRNPA2/B1 antibody were performed. The immunoprecipitated Circ-CDYL was determined by RT‒qPCR, followed by agarose gel electrophoresis.

(C–E) Exosomal Circ-CDYL levels were determined in the HCCLM3 cell line (C, left), Circ-CDYL-overexpressing HCC cells (SMMC-7721 Ov-Circ-CDYL) (D, left), and m⁶A sites-mutated Circ-CDYL-overexpressing HCC cells (SMMC-7721 Ov-Circ-CDYL^Mut) (E, left) after hnRNPA2/B1 interference (Si-hnRNPA2/B1). The ratio of cellular and exosomal Circ-CDYL to total cellular and exosomal RNA of the indicated cells, respectively, was calculated using 2^−ΔΔCT from RT‒qPCR (C–E, right). Ratios were normalized against β-actin and expressed as relative quantity with respect to negative control treatment set to a value of 1.